Novel Protein and method for producing the protein

ABSTRACT

A novel protein which binds to Osteoclastogenesis Inhibitory Factor (OCIF-binding molecule; OBM), a process for preparing the same, DNA encoding said protein, a protein having an amino acid sequence encoded by this DNA, a method for producing said protein by genetic engineering technique, and a pharmaceutical composition containing said protein.  
     Screening methods for a substance for controlling expression of said protein using said protein and the DNA, a substance which inhibits or modulates the biological activity of said protein, or a receptor which transmits the action of said protein through binding to said protein, the substance obtained by the screening methods, and a pharmaceutical composition which contains this substance.  
     An antibody for said protein, a process for preparing the same, a measuring method of said protein using the antibody, and a medicine comprising this antibody.

FIELD OF TECHNOLOGY

[0001] The present invention relates to a novel protein (OCIF-bindingmolecule, the protein may be hereinafter called OBM) which binds toosteoclastogenesis inhibitory factor (hereinafter it may be called OCIF)and a method to produce this protein.

[0002] The present invention also relates to DNA encoding this protein,proteins containing the amino acid sequence encoded by this DNA, amethod for the preparation of this protein utilizing genetic engineeringtechniques, and pharmaceutical compositions comprising this protein.

[0003] The present invention further relates to methods for screening,using this protein and the DNA, substances to control the expression ofthis protein, substances inhibiting or regulating the biologicalactivity of this protein, or receptors transducing the signal of theprotein by interacting with this protein, to substances obtained by thescreening, and to pharmaceutical compositions which comprise theresulting substances.

[0004] The present invention further relates to antibodies against thisprotein, methods for preparing the antibodies, and pharmaceuticalcompositions comprising these antibodies. Background ArtîH6,4400,839,5

[0005] Bone metabolism is dependent on the overall activity ofosteoblasts which control bone formation and osteoclasts which controlbone resorption. Abnormality of bone metabolism is considered to becaused by an imbalance of the bone formation and the bone resorption.Osteoporosis, hypercalcemia, Paget's disease, renal osteodystrophy,chronic rheumarthritis, osteoarthristis, and the like are known asdiseases accompanying abnormality of bone metabolism. Osteoporosis is atypical disease caused by such abnormality of bone metabolism. Thisdisease is generated when bone resorption by osteoclasts exceeds boneformation by osteoblasts. The disease is characterized by a decrease inboth the bone calcified material and the bone matrix. Although themechanism of this disease is not completely elucidated, the diseasecauses aches in bones, makes them fragile, and may result in fracturing.This disease is becoming a social problem because it increases thenumber of bedridden aged persons as the aged population becomes larger.Development of therapeutic agent for this disease is urgently desired.Disease due to a decrease in bone mass is expected to be cured bysuppressing bone resorption, accelerating bone formation, or improvingthe balance between bone resorption and formation. Bone formation isexpected to increase by accelerating proliferation, differentiation, oractivation of osteoblasts which form bone, or by suppressingproliferation, differentiation, or activation of osteoclasts whichresorb bone. In recent years, strong interest has been directed tohormones, low molecular weight substances, or physiologically activeproteins exhibiting such activities, and energetic basic research anddevelopment is underway on these subjects.

[0006] Drugs such as a calcitonin agents, active-form vitamin D₃ agents,hormone agents containing estradiol, ipriflavon, vitamin K₂, andbisphosphonate compounds have already been known as drugs to treat andshorten the treatment period of diseases related to bone. Clinical testsare in progress on active-form vitamin D₃ derivatives, estradiolderivatives, and bisphosphonate compounds of the second and the thirdgeneration to develop therapeutic agents with excellent efficacy andminimal side effects.

[0007] However, therapies using these agents were found not necessarilysatisfactory in terms of efficacy and therapeutic results. Developmentof novel therapeutic agents which are safer and with higher efficacy isurgently desired. Someagents used for the treatment of diseases relatedto bone metabolism are used only limitedly due to their side effects.Furthermore, treatments using two or more agents in combination arecurrently the mainstream in the treatment of diseases related to bonemetabolism such as osteoporosis. From such a point of view, developmentof drugs having action mechanisms different from those of conventionaldrugs, and exhibiting a higher efficacy and minimal side effects isdesired.

[0008] As mentioned above, the cells controlling bone metabolism areosteoblasts and osteoclasts. These cells are known to have close mutualinteractions called “coupling”. Specifically, cytokines such asInterleukins 1(IL-1), 3(IL-3), 6(IL-6), and 11(IL-11), granulocyticmacrophage-colony stimulating factor (GM-CSF), macrophage-colonystimulating factor (M-CSF), Interferon-γ (IFN-γ), tumor necrosis factorα (TNF-α), and transforming growth factory-β (TGF-β), secreted byosteoblastic stromal cells are known to accelerate or suppressdifferentiation or maturation of osteoclasts (Raisz: Disorders of Boneand Mineral Metabolism, 287-311, 1992; Suda et al.: Principles of BoneBiology, 87-102, 1996; Suda et al.: Endocrine Reviews, 4, 266-270, 1995,Lacey et al.: Endocrinology, 186, -369-2376, 1995). It has been reportedthat osteoblastic stromal cells play an important role in thedifferentiation and maturation of osteoclasts, as well as in osteoclastfunctions such as bone resorption by mature osteoclasts, throughcell-to-cell contact with immature osteoclast precursors or-matureosteoclasts.

[0009] A factor called osteoclast differentiation factor (ODF, Suda etal.: Endocrine Rev. 13, 66-80, 1992; Suda et al.: Bone 17, 87S-91S,1995) is thought to be expressed on the membrane of osteoblastic stromalcells and involved in the formation of osteoclasts through cell-to-cellcontact. According to this hypothesis, an ODF receptor is present in theprecursor cells of osteoclasts. However, so far neither the ODF nor thereceptor has been purified or identified. There are also no reportsrelating to their characteristics, action mechanism, or structure. Thus,the mechanism involved in differentiation and maturation of osteoclastshas not yet been sufficiently elucidated. Clarification of thismechanism will greatly contribute not only to the basic medicine, butalso to the development of novel drugs for the treatment of diseasesassociated with abnormality of bone metabolism.

[0010] The present inventors have conducted extensive studies in view ofthis situation and discovered an osteoclastogenesis inhibitory factor(OCIF) in a culture broth of human embryonic lung fibroblast, IMR-90(ATCC Deposition No. CCL186) (WO 96/26217).

[0011] The present inventors have been successful in cloning DNAencoding OCIF, production of recombinant OCIF in animal cells, andconfirmation of in vivo pharmaceutical effects (improving effect on bonemetabolism, etc.) of the recombinant OCIF. OCIF is expected to be usedas an agent for the prevention or treatment of diseases related toabnormality of bone metabolism, with higher efficacy than conventionaldrugs and less side effects.

DISCLOSURE OF THE INVENTION

[0012] The present inventors have searched for a protein which binds toosteoclastogenesis inhibitory factor (OCIF) and discovered that anOCIF-binding protein is specifically expressed on the osteoblasticstromal cells cultured in the presence of a bone resorption factor suchas active-form vitamin D₃ and parathyroid hormone (PTH). In addition,the present inventors have investigated the characteristics andphysiological functions of this OCIF-binding protein and found that theprotein exhibits biological activity of a factor which supports orpromotes the osteoclast differentiation and maturation from immatureprecursors of osteoclasts. These findings have led to the completion ofthe present invention. Further investigation into the protein of thepresent invention has proven that this is an important proteincontrolling the differentiation and maturation of osteoclasts fromimmature precursors of osteoclasts in a co-culture system of theosteoblastic stromal cells and spleen cells. The success inidentification and isolation of the protein which functions as a factorsupporting or promoting differentiation and maturation of osteoclasts inthe present invention has enabled screening for a novel medicine usefulfor abnormality of bone metabolism based on mechanism of bone metabolismutilizing the protein of the present invention.

[0013] Accordingly, an object of the present invention is to provide anovel protein (OCIF-binding molecule or OBM) which binds toosteoclastogenesis inhibitory factor (OCIF), and a method to producethis protein.

[0014] Another object of the present invention is to provide DNAencoding this protein, proteins containing an amino acid sequenceencoded by this DNA, a method for producing this protein utilizinggenetic engineering techniques, and pharmaceutical compositionscomprising this protein.

[0015] A further object of the present invention is to provide methodsfor screening substances which control expression of this protein usingthis protein and the DNA, substances inhibiting or regulating thebiological activity of this protein, receptors transducing the action ofthe protein by binding to the protein, substances obtained by thescreening, and pharmaceutical compositions which comprises thesesubstances.

[0016] A still further object of the present invention is to provideantibodies against this protein, methods for preparing the antibodies,and pharmaceutical compositions comprising these antibodies.

[0017] The protein of the present invention has the followingphysicochemical properties and biological activity.

[0018] (a) Affinity: specifically binds to the osteoclastogenesisinhibitory factor (OCIF) and exhibits high affinity to OCIF(dissociation constant on cell membrane: Kd=10⁻⁹ M or less);

[0019] (b) Molecular weight: has a molecular weight of approximately30,000-40,000 when determined by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) under non-reducing conditions and an apparent molecularweight of approximately 90,000-110,000 when cross-linked to a monomerform OCIF; and

[0020] (c) Biological activity: exhibits activity supporting orpromoting osteoclast differentiation and maturation in a co-culturesystem of the mouse osteoblastic stromal cells and mouse spleen cells inthe presence of bone resorption factors such as active-form vitamin D₃and parathyroid hormone (PTH).

[0021] A co-culture system of ST2, a mouse osteoblastic stromal cellline, and mouse spleen cells in the presence of active-form vitamin D₃or PTH is well known as a typical in vitro culture system for osteoclastformation. The cells expressing the protein of the present invention canbe determined by testing the binding of OCIF to mouse osteoblasticstromal cells or mouse spleen cells cultured in the presence or absenceof active-form vitamin D₃. The protein of the present invention isspecified as the protein which is induced specifically on theosteoblastic stromal cells cultured in the presence of an osteotropicfactor such as active-form vitamin D₃ or PTH. In addition, the proteinof the invention can be specified as a protein exhibiting biologicalactivity supporting or promoting differentiation and maturation ofosteoclasts from the following results. That is, the osteoclastformation is inhibited dose dependently by the addition of 1 to 40 ng/mlof OCIF to the above-mentioned co-culture system in the presence of theactive-form vitamin D₃, the time course of expression of the protein ofthe present invention on ST2 cells in the presence of active-formvitamin D₃ well correlates with the time course of osteoclast formationin the co-culture. In addition, the amount of protein of the presentinvention expressed on ST2 cells correlates with the capability of thecells to support the osteoclast formation, and the binding of OCIF tothe protein of the present invention on the ST2 cells completelysuppresses osteoclasts formation.

[0022] The affinity of the protein of the present invention to OCIF canbe evaluated by labeling OCIF and examining the binding of the labeledOCIF to the surface of animal cell membrane. OCIF can be labeled by aconventional protein-labeling method such as radioisotope or fluorescentlabeling. Labeling of tyrosine residues with ¹²⁵I can be given as aspecific example of labeling of the OCIF with an radioisotope. Labelingmethods such as iodogen method, chloramine T method, and enzymaticmethod can be utilized. The binding of the labeled OCIF to the surfacemembrane of animal cell can be tested by a conventional method. Theaddition of unlabeled OCIF to the medium used for the binding assay to aconcentration, 100 to 400 times the concentration of labeled OCIF,ensures measurement of non-specific binding. The amount of specificbinding of OCIF can be calculated by subtracting the amount ofnon-specific binding from the total amount of binding of the labeledOCIF. The affinity of the protein of the present invention expressed onthe cell membrane to OCIF can be evaluated by changing the amount oflabeled OCIF and analyzing the specific binding by Scatchard plot.

[0023] The determined affinity of the protein of the present inventionto OCIF is approximately 100-500 pM. The protein of the presentinvention is specified by a high affinity (dissociation constant on cellmembrane: Kd=10⁻⁹ M or less) to osteoclastogenesis inhibitory factor(OCIF). The molecular weight of OBM can be accessed by gel filtrationchromatography, SDS-PAGE, or the like. SDS-PAGE is preferred in order toaccurately determine the molecular weight. The OBM is specified as aprotein having a molecular weight of approximately 40,000 (40,000±4,000)under reducing conditions.

[0024] The protein of the present invention can be obtained from mouseosteoblastic stromal cell line, ST2, mouse preadipocyte cell line, PA6,human osteoblastic cell lines, or other osteoblastic cells selected frommammalians such as humans, mice, or rats. As the substances to induceexpression of the protein of the present invention, osteotropic factorssuch as active-form vitamin D₃ (calcitriol), parathyroid hormone (PTH),interleukin (IL)-1, IL-6, IL-11, Oncostatin M, and leukemia inhibitoryfactor (LIF) can be given. These substances can be added in theconcentration of 10⁻⁸ M (active-form vitamin D₃ and PTH), 10 ng/ml(IL-11), or 1 ng/ml (Oncostatin M). IL-6 is preferably used at aconcentration of 20 ng/ml with 500 ng/ml soluble IL-6 receptor.Preferably, confluent cells of mouse osteoblastic stromal cell line,ST2, cultured in α-MEM medium to which 10⁻⁸ M of active-form vitamin D₃,10⁻⁷ M of dexamethasone, and 10% fetal bovine serum were added can beused. The cultured cells may be collected by scraping with a cellscraper. The collected cells may be stored at −80° C. until use.

[0025] The protein of the present invention can be purified efficientlyfrom the membrane fractions of the collected cells. The membranefractions can be prepared by a conventional method which is used toprepare intracellular organella. Various types of protease inhibitorsmay be added to the buffer solution used for the preparation of themembrane fractions. Examples of the protease inhibitors include serineprotease inhibitors, thiol protease inhibitors, and metaproteaseinhibitors. PMSF, APMSF, EDTA, o-phenanthroline, leupeptine, pepstatinA, aprotinin, soybean trypsin inhibitor are givens as specific examples.A Daunce homogenizer, a polytron homogenizer, or a ultrasonic processorcan be used to homogenize the cells. The cell homogenate is suspended ina buffer solution containing 0.5 M of sucrose and centrifuged for 10minutes at 600×g, to separate the nucleus and undisrupted cells asprecipitate. The supernatant is centrifuged for 90 minutes at 150,000×gto obtain a membrane fractions as precipitate. The obtained membranefraction is treated by various types of detergents to efficientlysolubilize and extract the protein of the present invention from thecell membrane. Detergents which are commonly used to solubilize cellmembrane proteins, such as CHAPS(3-[(3-cholamidopropyl)-dimethylamonio]-1-propanesulfonate), TritonX-100, Nikkol, and n-octyl glycoside, can be used. Preferably, 0.5%CHAPS is added to the membrane fraction and the mixture is stirred for 2hours at 4° C. to solubilize the protein of the present invention. Thesample thus prepared is centrifuged at 150,000×g for 60 minutes toobtain the solubilized membrane fraction as a supernatant.

[0026] The protein of the present invention can be purified from thesolubilized membrane fraction with a column, gel, or resin coupled withOCIF. The immobilized OCIF may be that isolated from a culture broth ofhuman embryonic lung fibroblasts, IMR-90, described in WO 96/26217 orrOCIF prepared using genetic engineering techniques. rOCIF can beprepared by introducing human cDNA, mouse cDNA, or rat cDNA into anexpression vector according to a conventional method, transducing theconstructed vector in animal cells such as CHO cells, BHK cells, orNamalwa cells, or in insect cells to produce rOCIF, and purifying rOCIF.Obtained OCIF has a molecular weight of approximately 60 kDa(monomer-form) or 120 kDa (dimer-form). The dimer-form OCIF ispreferable for immobilization. Given as examples of the gels and resinsto which OCIF is immobilized are ECH Sepharose 4B, EAH Sepharose 4B,Thiopropyl Sepharose 6B, CNBr-activated Sepharose 4B, activated CHSepharose 4B, Epoxy activated Sepharose 6B, activated thiol Sepharose 4B(these are manufactured by Pharmacia Co.), TSKgel AF-Epoxy Toyopal 650,TSKgel AF-Amino Toyopal 650, TSKgel AF-Formyl Toyopal 650, TSKgelAF-Carboxy Toyopal 650, TSKgel AF-Tresyl Toyopal650 (these aremanufactured by Tosoh Corporation), Amino-Cellulofine,Carboxy-Cellulofine, FMP activated Cellulofine, Formyl-Cellulofine(these are manufactured by Seikagaku Kogyo Co.), Affigel 10, Affigel 15,and Affiprep 10 (these are manufactured by BioRad Co.). As columns towhich OCIF is immobilized, HiTrap NHS-activated column (Pharmacia Co.),TSKgel Tresyl-5PW (Tosoh Corporation), etc. can be given. As a specificexample of the method for immobilizing OCIF to a HiTrap NHS-activatedcolumn (1 ml, Pharmacia Co.), the following method can be given.Specifically, 1 ml of 0.2M NaHCO₃/0.5 M NaCl solution (pH 8.3)containing 13.0 mg of OCIF is injected to the column to perform couplingreaction at room temperature for 30 minutes. 0.5 M ethanolamine/0.5 MNaCl (pH 8.3) and 0.1 M acetic acid/0.5 M NaCl (pH 4.0) are sequentiallyapplied to the column. Then, the column is again washed with 0.5 Methanolamine/0.5 M NaCl (pH 8.3) and the column is allowed to stand forone hour at room temperature to block excess active groups. The columnis sequentially washed twice with 0.5 M ethanolamine/0.5 M NaCl (pH 8.3)and 0.1 M acetic acid/0.5 M NaCl (pH 4.0), and then washed with 50 mMTris/1M NaCl/0.1% CHAPS solution (pH 7.5), thereby obtaining aOCIF-immobilized column. The protein of the present invention can beefficiently purified by a OCIF-immobilized column prepared in thismanner, or an OCIF-immobilized gel or resin.

[0027] It is desirable to add the various above-mentioned proteaseinhibitors to the buffer solutions used for the purification of theprotein to suppress degradation of the protein of the present invention.The protein of the present invention can be purified by loading theabove-mentioned solubilized membrane fraction on the OCIF-immobilizedcolumn or by mixing with the OCIF-immobilized gel or resin, and elutingthe protein from the column, gel, or resin with acid, various proteindenaturing agents, cacodylate buffer, and the like. It is desirable touse an acid for elution and to neutralize immediately after elution tominimize denaturation of the protein of the present invention. As theacidic solution used for elution, 0.1 M glycine-hydrochloric acidsolution (pH 3.0), 0.1 M glycine-hydrochloric acid solution (pH 2.0),0.1 M sodium citrate solution (pH 2.0), and the like can be given.

[0028] The protein of the present invention can be further purified byconventional purification methods used for purification of variousproteins from biological materials and by various purification methodsutilizing the physicochemical properties of this protein. To concentratesolutions containing the protein of the present invention, conventionaltechniques used in the purification process for proteins such as ultrafiltration, freeze drying, and salting-out, can be used. Ultrafiltration with Centricon-10 (BioRad Co.), for example, is preferablyused. As a means for the purification, various techniques conventionallyutilized for the purification of proteins, such as ion exchangechromatography, gel filtration chromatography, hydrophobicchromatography, reverse phase chromatography, and preparativeelectrophoresis, are used in combination. More specifically, it ispossible to purify the protein of the present invention by a combinationof gel filtration chromatography with Superose 12 column (Pharmacia Co.)and reverse phase chromatography. To detect the protein of the presentinvention in the purification process, the binding activity of theprotein of the present invention to the immobilized OCIF is examined orthe material bound to the immobilized OCIF is immuno precipitated withan anti-OCIF antibody and analyzed by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE).

[0029] The obtained protein of the present invention is useful as anagent for treating diseases caused by abnormality of bone metabolismsuch as osteopetrosis or as a reagent for research and diagnosis ofthese diseases.

[0030] The present invention further provides DNA encoding a novelprotein (OCIF-binding molecule or OBM) which binds to osteoclastogenesisinhibitory factor, proteins containing the amino acid sequence encodedby this DNA, a method for the preparation of this protein by the geneticengineering technique, and pharmaceutical compositions comprising thisprotein. Furthermore, the present invention provides methods forscreening substances to regulate expression of OBM, a method forscreening substances inhibiting or modifying the biological activity ofOBM, or a method for screening receptors transducing the action of OBMby binding to OBM, and pharmaceutical compositions which comprisessubstances obtained as a result of the screening.

[0031] The novel protein OBM which is encoded by the DNA of the presentinvention has the following physicochemical properties and biologicalactivity.

[0032] (a) binds specifically to osteoclastogenesis inhibitory factor(OCIF),

[0033] (b) has a molecular weight of approximately 40,000 (±4,000) whendetermined by SDS-PAGE under reducing conditions and an apparentmolecular weight of approximately 90,000-110,000 when crosslinked tomonomer-form OCIF, and

[0034] (c) exhibits activity supporting or promoting differentiation andmaturation of osteoclasts.

[0035] Human osteoclastogenesis inhibitory factor (OCIF) which is usedas a probe to identify the DNA encoding OBM, the OCIF-binding moleculeof the present invention, and to evaluate properties of OBM can beisolated from a culture broth of a human embryonic lung fibroblast cellline, IMR-90, according to WO No. 96/26217. Recombinant human OCIF,recombinant mouse OCIF, recombinant rat OCIF, and the like can also beused for the isolation and identification of the DNA coding OBM. Theserecombinant OCIF proteins can be produced by inserting DNA fragmentsencoding these proteins into expression vectors according toconventional methods, expressing in animal cells such as CHO cells, BHKcells, or Namalwa cells, or in insect cells, and purifying them.

[0036] As a method for isolating cDNA encoding a target protein (cDNAcloning), the method comprising determination of a partial amino acidsequence of the protein and isolation of the target cDNA byhybridization utilizing the nucleotide sequence corresponding to theamino acid sequence can be employed. Another available method, even inthe case where the amino acid sequence of the protein is not known,comprises constructing a cDNA library in a expression vector,introducing the cDNA into cells, and screening for the expression of thetarget protein to isolate the objective cDNA (expression cloning method,D'Andrea et al.: Cell 57, 277-285, 1989; Fukunaga et al.: Cell61,341-350, 1990). In the expression cloning method, suitable host cellssuch as bacteria, yeast, animal cells, and the like are selecteddepending on the objective. In many cases, animal cells are selected asthe host cells for cloning cDNA encoding a protein such as the proteinof the present invention which is considered to be present in animalcell membrane surface. Normally, host cells showing high efficiency forDNA transfection and achieving expression of the introduced DNA at highlevels are selected. One of such animal cells is the monkey kidneycells, COS-7, used in the present invention. Because SV40 large Tantigen is expressed in the COS-7 cell, a plasmid which has a replicatorof SV40 can be present as an episome of multiple copies in the cell, sothat a high level of expression is expected. In addition, becauseexpression of a target protein by COS-7 cells reaches a maximum within afew days after introduction of DNA, the cell is suitable for rapidscreening. A combination of this host cell with a plasmid capable ofhigh expression ensures gene expression of an extremely high level. Thefactor exhibiting the greatest influence on the expression of a gene ona plasmid is a promoter. Promoters such as SRα promoter andcytomegalovirus-derived promoters are used as high expression promoters.To screen for the cDNA encoding a membrane protein by the expressioncloning strategy, screening procedures such as binding method, panningmethod, or film emulsion method are used.

[0037] The present invention relates to DNA encoding the protein (OBM)which specifically binds to OCIF, isolated by the combination of theexpression cloning strategy and the screening by the binding method, tothe expressed protein, and to screening of physiologically activesubstances using the DNA or the expressed protein. OBM encoded by theDNA of the present invention can be detected by labeling OCIF andtesting the binding of the labeled OCIF to membrane surface of an animalcell. OCIF can be labeled by a conventional labeling method such asradioisotope labeling method or fluorescent labeling method which isused for labeling common proteins. Labeling tyrosine residues by ¹²⁵Ican be given as a specific example of labeling OCIF with a radioisotope.Labeling methods such as the iodogene method, chloramine T method, andenzymatic method can be utilized. The binding of labeled OCIF to theanimal cell membrane surface can be tested by conventional methods. Theaddition of unlabeled OCIF to the medium used for the test to aconcentration, 100 to 400 times the concentration of labeled OCIF,enables quantification of the amount of non-specific binding. The amountof specific binding of OCIF can be calculated by subtracting the amountof non-specific binding from the total amount of binding of the labeledOCIF.

[0038] The present inventors assumed that there is interaction betweenthe factor involved in differentiation of osteoclasts and OCIF. Based onthis assumption, to isolate the protein to which recombinant OCIF binds,the inventors screened the expression library prepared from mRNA ofmouse osteoblastic stromal cell line, ST2, according to the followingmethod. Specifically, DNA synthesized using ST2 mRNA was inserted intoan expression vector for animal cells and the vector with the insert wasintroduced into monkey kidney COS-7 cells. The objective proteinexpressed on the COS-7 cells was screened using OCIF labeled with ¹²⁵Ias a probe. As a result, DNA encoding the protein which bindsspecifically to OCIF was isolated. The nucleotide sequence of the DNAencoding this OCIF-binding molecule (OCIF-binding molecule; OBM) wasthen determined. Moreover, OBM encoded by this DNA was found to bindspecifically and strongly to OCIF, on the cell membrane.

[0039] Comparatively mild conditions for hybridization of DNA in thepresent invention are the conditions, for example, wherein DNA istransferred to a nylon membrane and immobilized thereto according toconventional methods and hybridized in a buffer solution forhybridization with a probe DNA labeled with an isotope at a temperatureof 40-70° C. for about 2 hours to overnight, followed by washing in0.5×SSC (0.075 M sodium chloride and 0.0075 M sodium citrate)at 45° C.for 10 minutes. Specifically, Highbond N (Amersham Co.) is used as thenylon membrane to transfer and immobilize DNA thereon. DNA is thenhybridized with a probe DNA labeled with ³²P in a rapid hybridizationbuffer (Amersham Co.) at 65° C. for 2 hours, followed by washing with0.5×SSC (0.075 M sodium chloride and 0.0075 M sodium citrate) at 45° C.for 10 minutes.

[0040] A co-culture system of mouse osteoblastic stromal cells, ST2, andmouse spleen cells in the presence of active-form vitamin D₃ or PTH iswell known as a typical in vitro culture system forosteoclast-formation. The protein of the present invention is specifiedas the protein which is induced specifically on the osteoblastic stromalcells cultured in the presence of an agent which accelerates boneresorption such as active-form vitamin D₃ or PTH. In addition, becauseof the fact that formation of osteoclasts is stimulated by the additionof the protein encoded by the DNA of the present invention to mousespleen cells cultured even in the absence of active-form vitamin D₃ orPTH, OBM which is encoded by the DNA of the present invention isconsidered to be involved in the differentiation and maturation ofosteoclasts

[0041] Recombinant OBM can be produced by inserting the DNA of thepresent invention into an expression vector to construct a plasmid andintroducing the plasmid into various cells or microorganisms to expressrecombinant OBM. As a host in which recombinant OBM is expressed,mammalianian cells such as COS-7, CHO, Namalwa, or bacteria such asEscherichia coli can be used. OBM may be expressed as amembrane-bound-form protein using the full length DNA or as asecretion-form or a soluble-form protein by removing the portionencoding the transmembrane domain. The produced recombinant OBM can beefficiently purified using a suitable combination of conventionalpurification methods used for common proteins, such as affinitychromatography using OCIF-immobilized columns, ion exchangechromatography, and gel filtration chromatography. The obtained proteinof the present invention is useful as an agent for treating diseasescaused by abnormality of bone metabolism such as osteopetrosis or as areagent for research and diagnosis of such diseases.

[0042] The following screening operations can be carried out using theprotein OBM encoded by the DNA of the present invention: (1) screeningof substances which regulate expression of OBM, (2) screening ofsubstances which specifically bind to OBM and inhibit the biologicalactivity of OBM, and (3) screening of proteins which are present inosteoclast precursor cells and transduce the biological activity of OBM(OBM receptor). It is also possible to develop antagonists and agonistsusing this OBM receptor. In the combinatorial chemistry using theabove-mentioned OBM or OBM receptor, a peptide library used for thescreening of the antagonists or agonists can be prepared by thefollowing method. Specifically, one of the methods is split method (Lamet al.; Nature 354, 82-84, 1991). According to this method, syntheticcarriers (beads) each comprising a specific amino acid (unit) boundthereto are prepared separately for all units. The synthesized carriersare mixed altogether and divided into portions equal to the number ofthe units. Then, the next units are bound. This procedure is repeated“n” times to produce a library containing carriers to which “n” unitsare bound. According to this synthetic method, each carrier pool has onetype of sequence. Therefore, it is possible to identify a peptidespecifically binding to the protein of the present invention byselecting the pool which gives a signal positive in this screeningmethod using the protein of the present invention, and determining theamino acid sequence of the peptide bound on the pool. Another method isphage display method which utilizes phage carrying synthetic DNA whichencode peptides with random amino acid sequences. The method has theadvantage of increasing the number of molecules in the library ascompared with the above-mentioned synthetic peptide library method, buthas the disadvantage of less variety for a given number of moleculesbecause there can be particular sequences which are missing in thelibrary if the phages are unable to express those sequences. In thephage display method, the screening system using the protein of thepresent invention can also be applied to determine the nucleotidesequence encoding the peptide. That is, the phage specifically bindingto the protein of the present invention is concentrated by panning, theselected phage is amplified in Escherichia coli, and the nucleotidesequence encoding the peptide is determined. In addition, a peptideexhibiting high specificity and high affinity to OBM or OBM receptor canbe screened from a peptide library using the screening systems mentionedabove in (2) and (3) by screening in the presence of OBM or OCIF whileincreasing the concentration of OBM or OCIF. Only positive carrier poolsor phages are selected in this manner. For example, low molecular weightpeptide agonists exhibiting an EPO (erythropoietin)-like activity werescreened from a peptide library using a receptor of erythropoietin (EPO)which is a hematopoietic hormone, the tertiary structure of thissubstance was analyzed, and based on this tertiary structure, lowmolecular weight substances (antagonist) exhibiting the EPO-likeactivity were synthesized (Nicholas et al.: Science, 273, 458-463,1996).

[0043] The present inventors have previously discovered using theosteoclastogenesis inhibitory factor, OCIF, that an OCIF-binding proteinis specifically expressed on osteoblastic stromal cell line, ST2,cultured in the presence of a osteotropic factor such as active-formvitamin D₃ or parathyroid hormone (PTH). The inventors further foundthat this protein exhibits a biological activity to support or stimulatedifferentiation or maturation of osteoclasts from immature osteoclastprecursor cells, and clarified various physicochemical properties andthe biological activity of this protein by purification thereof. Inorder to compare the recombinant OBM expressed by the DNA of the presentinvention and the above-mentioned purified natural type protein whichspecifically binds to OCIF, the present inventors investigated thephysicochemical properties and biological activities of the twoproteins. As a result, the two proteins were confirmed {circle over (1)}to be both membrane-bound proteins which specifically bind to OCIF,{circle over (2)} to have molecular weights of approximately 40,000determined by SDS-PAGE, and {circle over (3)} to have apparent molecularweights of about 90,000-110,000 when cross-linked to a monomer formOCIF. Not only are these physicochemical properties identical, but bothproteins exhibit a biological activity to support or stimulatedifferentiation or maturation of osteoclasts, suggesting the possibilitythat these are the same protein. In addition, a rabbit anti-OBMpolyclonal antibody produced using the purified protein prepared byexpressing the DNA of the present invention by a genetic engineeringtechnique (recombinant OBM) was confirmed to cross react with theabove-described purified natural type protein, to inhibit specificbinding of this purified natural type protein and OCIF in the samemanner as the antibody inhibits specific binding of OBM and OCIF. Basedon these results, it is clear that the recombinant OBM expressed by theDNA of the present invention is identical to the natural type proteinwhich specifically binds to OCIF.

[0044] To isolate a gene (cDNA) encoding human OCIF-binding protein(hereinafter called human OBM) which specifically binds to OCIF andexhibits the activity to support and stimulate differentiation andmaturation of osteoclasts from mouse spleen cells in the same manner asthe natural type or recombinant mouse OBM dose, a cDNA library preparedfrom mRNA derived from human lymph nodes was screened using a human OBMcDNA fragment as a probe. The human OBM cDNA fragment was obtained bypolymerase chain reaction (PCR) in accordance with the method mentionedabove using both cDNA prepared from human lymph node as a template andthe primer which was prepared from mouse OBM cDNA. As a result, cDNAencoding the human protein which specifically binds to OCIF was isolatedand the nucleotide sequence of the cDNA encoding this human OCIF-bindingprotein molecule (i.e. the cDNA encoding human OBM) was determined.Similar to mouse OBM, this human OBM encoded by the cDNA hascharacteristics to bind to OCIF strongly and specifically on the cellmembrane and exhibits the activity to support and promotedifferentiation and maturation of osteoclasts from mouse spleen cells.Specifically, the present invention provides DNA encoding novel humanOBM protein which binds to osteoclastogenesis inhibitory factor (OCIF),a protein which possesses the amino acid sequence encoded by the DNA, amethod for producing the protein exhibiting characteristics ofspecifically binding to OCIF and the activity to support and promotedifferentiation and maturation of osteoclasts from mouse spleen cells bygenetic engineering techniques, pharmaceutical compositions comprisingthis protein for the treatment of diseases caused by abnormality of bonemetabolism, a method for screening substances regulating expression ofhuman OBM, a method for screening substances which inhibit or modulatethe activity of human OBM by binding to it, a method for screeningreceptors which bind to human OBM and transmit the action of OBM, and apharmaceutical compositions comprising the substances obtained by thesescreenings.

[0045] The present invention further provides DNA encoding novel humanOBM protein which specifically binds to OCIF and exhibits the biologicalactivity to support and promote differentiation and maturation ofosteoclasts, a protein which possesses the amino acid sequence encodedby the DNA, a method for producing the protein exhibitingcharacteristics of specifically binding to OCIF and the activity tosupport and promote differentiation and maturation of osteoclasts bygenetic engineering techniques, and pharmaceutical compositionscomprising this protein for the treatment of diseases causingabnormality of bone metabolism. Furthermore, the present inventionprovides a method for screening substances regulating expression ofhuman OBM, a method for screening substances which inhibit or modulatethe activity of human OBM by binding to it, a method for screeningreceptors binding to human OBM and transmitting the action of OBM,antibodies against human OCIF binding protein, and pharmaceuticalcompositions comprising these antibodies for the prevention or treatmentof diseases causing abnormality of bone metabolism.

[0046] The novel, human OCIF-binding protein molecule (OBM) which isencoded by the DNA of the present invention has the followingphysicochemical properties and biological activity.

[0047] (a) binds specifically to osteoclastogenesis inhibitory factor(OCIF) (WO 96/26217),

[0048] (b) has a molecular weight of approximately 40,000 (±5,000) whendetermined by SDS-PAGE under reducing conditions and an apparentmolecular weight of approximately 90,000-110,000 when crosslinked with amonomer form OCIF, and

[0049] (c) exhibits activity to support and stimulate differentiationand maturation of osteoclasts.

[0050] Mouse OBM cDNA which encodes mouse OCIF-binding protein and usedas a probe to isolate and identify the cDNA encoding human OBM of thepresent invention, can be isolated according to the above-mentionedmethod from a cDNA library of mouse osteoblastic stromal cell line, ST2.Human osteoclastogenesis inhibitory factor (OCIF) which is necessary toevaluate the properties and the biological activity of the proteinobtained by expression of human OBM cDNA, can be prepared according tothe method described in WO 96/26217 by isolating from a culture broth ofhuman fibroblast cell line, IMP-90, or by genetic engineering techniquesusing the DNA encoding OCIF. Recombinant human OCIF, recombinant mouseOCIF, recombinant rat OCIF, or the like can be used for the assessmentof the properties and biological activity of human OBM. Theserecombinant OCIF can be obtained according to conventional methods byinserting cDNA into an expression vector, expressing the cDNA in animalcells such as CHO cells, BHK cells, or Namalwa cells, or in insectcells, and purifying the expressed proteins.

[0051] The following methods can be used to isolate human cDNA encodingthe target protein (cDNA cloning). {circle over (1)} A method comprisingpurifying the protein, determining the partial amino acid sequence ofthe protein, and isolating the target cDNA by hybridization using theDNA fragment comprising nucleotide sequence corresponding to the aminoacid sequence as a probe, {circle over (2)}a method applied even in thecase where the amino acid sequence of the protein is not known, whichcomprises constructing a cDNA library in a expression vector,introducing the cDNA library into cells, and screening for theexpression of the target protein to isolate the objective cDNA(expression cloning method), and {circle over (3)} a method of isolatingcDNA encoding the target human protein from the cDNA library constructedusing human cells or tissues by hybridization or by the use ofpolymerase chain reaction (PCR) using the cDNA encoding the protein ofmammalian origin (other than human) which possesses the samecharacteristics and biological activity as the target protein of humanorigin as a probe, assuming that the cDNA probe has high homology withthe human-origin cDNA which to be cloned. Based on the assumption thathuman OBM cDNA has a high homology with mouse OBM cDNA, it is possibleto determine which cells or tissues produce human OBM by Northernhybridization method using the mouse OBM cDNA as a probe. Human OBM cDNAcan be obtained by the following method using the mouse OBM primerprepared from the mouse OBM cDNA. Human OBM cDNA fragments can beprepared by the PCR method using cDNA prepared from human OBM-producingtissues such as human lymph nodes as a template. These human OBM cDNAfragments are used as probes for screening the cDNA library of humanOBM-producing cells or tissues which were identified according to themethod mentioned above. The present invention relates to the DNAencoding human OBM which has characteristics of specific binding to OCIFand exhibits activity to support and promote differentiation andmaturation of osteoclasts. Because the OBM which is encoded by the DNAof the present invention is a membrane-bound type protein whichcomprises a transmembrane domain, this protein can be detected bylabeling OCIF and by examining the binding of the labeled OCIF to thesurface of animal cells in which the cDNA of the present invention wasexpressed. The above-described labeling method using radioisotope orfluoresceine conventionally applied to labeling proteins can be used forlabeling OCIF.

[0052] The molecular weight of the protein expressed by the human OBMcDNA of the present invention can be accessed by gel filtrationchromatography, SDS-PAGE, or the like. In order to accurately determinethe molecular weight, it is desirable to use the SDS-PAGE method, bywhich human OBM was specified as a protein having a molecular weight ofapproximately 40,000 (40,000±5,000) under reducing conditions.

[0053] Comparatively mild conditions for hybridization of DNA in thepresent invention are the conditions, for example, wherein DNA istransferred to a nylon membrane and immobilized thereto according to aconventional method and hybridized with a probe DNA labeled with anisotope in a buffer solution for hybridization at a temperature of40-70° C. for about 2 hours to overnight, followed by washing in 0.5×SSC(0.075 M sodium chloride and 0.0075 M sodium citrate)at 45° C. for 10minutes. Specifically, Highbond N (Amersham Co.) is used as the nylonmembrane to transfer and immobilize DNA thereon. The DNA is thenhybridized with a probe DNA labeled with ³²P in a rapid hybridizationbuffer (Amersham Co.) at 65° C. for 2 hours, followed by washing with0.5×SSC at 45° C. for 10 minutes.

[0054] A co-culture system of mouse osteoblastic stromal cells, ST2, andmouse spleen cells in the presence of active-form vitamin D₃ or PTH iswell known as a typical in vitro culture system forosteoclast-formation. Interaction by adhesion of osteoblastic stromalcells and spleen cells and presence of an osteotropic factor such asactive-form vitamin D₃ or PTH are indispensable for the osteoclastsformation in this in vitro culture system. In this in vitro culturesystem, COS cells, monkey kidney cells having no osteoclastformation-supporting capability, acquire capability to supportosteoclasts formation from spleen cells in the absence of an osteotropicfactor when the cDNA of the present invention was expressed asosteoblastic stromal cell line ST2 did. Based on the fact that the cDNAof the present invention encodes a protein comprising a transmembranedomain form, this cDNA can be expressed as a secretion form orsoluble-form by removing the part which encodes this transmembranedomain. It was confirmed that osteoclasts can be formed by the additionof the secretion form human OBM to the above-mentioned in vitro culturesystem in the absence of osteotropic factors. Based on these results,the human OBM which is encoded by the cDNA of the present invention isspecified as the factor involved in the differentiation and maturationof osteoclasts.

[0055] A recombinant human OBM can be prepared by inserting the cDNA ofthe present invention into an expression vector, preparing a human OBMexpression plasmid, introducing the plasmid into various cell strainsand expressing OBM in the cells. Mammalianian cells such as COS-7, CHO,Namalwa cells, or bacteria such as Escherichia coli can be used as ahost for expressing OBM. In this case, OBM may be expressed as amembrane-bound-form protein, using the full length DNA, or as asecretion-form or soluble-form protein by removing the part encoding thetransmembrane domain. The recombinant OBM thus produced can beefficiently purified using a suitable combination of conventionalpurification methods used for common proteins such as affinitychromatography using OCIF immobilized columns, ion exchangechromatography, and gel filtration chromatography. Human OBM of thepresent invention thus obtained is useful as an agent for treatingdiseases caused by abnormality of bone metabolism such as osteopetrosisor as a reagent for research and diagnosis of such diseases.

[0056] The following screening operations can be carried out using theprotein OBM encoded by the DNA of the present invention: (1) screeningof substances which can regulate expression of human OBM, (2) screeningof substances which specifically bind to human OBM and inhibit or modifythe biological activity of OBM, and (3) screening of human proteinswhich are present in osteoclast precursor cells and transmit thebiological activity of human OBM (human OBM receptor). It is alsopossible to develop antagonists and agonists using this human OBMreceptor. In the combinatorial chemistry using the human OBM or humanOBM receptor, peptide libraries required for the screening ofantagonists or agonists can be produced by the same method as used forthe screening using mouse OBM. A peptide with extremely high specificityand affinity can be obtained by screening peptide libraries using humanOBM instead of mouse OBM.

[0057] Although this OBM is very useful as mentioned above andantibodies specifically recognizing OBM and enzyme immunoassay usingthese antibodies are indispensable in determination of OBMconcentration, no antibodies useful for the access of OBM concentrationhave been so far available. In addition, an anti-OBM antibody oranti-sOBM antibody which neutralizes the biological activity of OBM orsOBM is supposed to suppress the activity of OBM or sOBM, specificallythe activity to induce osteoclasts formation. These are expected to beuseful as therapeutic agents to treat abnormality of bone metabolism.However, no such antibodies have so far been available.

[0058] In view of this situation, the present inventors have conductedextensive studies. As a result, the present inventors have foundantibodies (anti-OBM/sOBM antibodies) which recognize both OBM, amembrane-bound protein which specifically binds to osteoclastogenesisinhibitory factor (OCIF), and soluble OBM (sOBM) which lack atransmembrane domain. Accordingly, the present invention providesantibodies (anti-OBM/sOBM antibodies) which recognizes both OBM, amembrane-bound protein which specifically binds to osteoclastogenesisinhibitory factor (OCIF), and sOBM which lack a transmembrane domain; amethod for the preparation thereof; a method for determination of OBMand sOBM concentrations using these antibodies; and agents for theprevention or treatment of diseases resulting from abnormality of bonemetabolism.

[0059] The present invention relates to antibodies (anti-OBM/sOBMantibodies) which recognize both the OBM, a membrane-bound protein whichspecifically binds to osteoclastogenesis inhibitory factor (OCIF), andsoluble OBM (sOBM) which lack a transmembrane domain; a method for thepreparation thereof;a method for quantifying OBM and sOBM using theseantibodies; and agents for the prevention or treatment of diseasesresulting from abnormality of bone metabolism.

[0060] The antibodies of the present invention exhibit activity ofneutralizing the osteoclastogenesis accelerating activity which is thebiological activity of OBM and sOBM and comprises the antibodies havingthe following characteristics:

[0061] (a) polyclonal antibody which recognizes both mouse OBM and mousesOBM (anti-mouse OBM/sOBM polyclonal antibody),

[0062] (b) polyclonal antibody which recognizes both human OBM and humansOBM (anti-human OBM/sOBM polyclonal antibody),

[0063] (c) monoclonal antibodies which recognizes both mouse OBM andmouse sOBM (anti-mouse OBM/sOBM monoclonal antibodies),

[0064] (d) monoclonal antibodies which recognize both human OBM andhuman sOBM (anti-human OBM/sOBM monoclonal antibodies), and

[0065] (e) anti-human OBM/sOBM monoclonal antibodies which cross reactto both mouse OBM and mouse sOBM.

[0066] The polyclonal antibody which recognizes both mouse OBM and mousesOBM (hereinafter called anti-mouse OBM/sOBM polyclonal antibody) andthe polyclonal antibody which recognizes both human OBM and human sOBM(hereinafter called anti-human OBM/sOBM polyclonal antibody) wereproduced by the following method. The purified mouse OBM used as anantigen for immunization can be obtained according to theabove-mentioned method. Especially, mouse osteoblastic stromal cellline, ST2, was treated with active-form vitamin D₃, and OBM on the cellmembrane was purified using an OCIF-immobilized column and gelfiltration chromatography, thereby obtaining natural mouse -OBM (nativeOBM). The above-mentioned mouse OBM cDNA (Sequence Table, Sequence No.15) or human OBM cDNA (Sequence Table, Sequence ID No. 12) was insertedinto an expression vector according to conventional methods. Recombinantmouse OBM (Sequence Table, Sequence ID No. 1) and recombinant human OBM(Sequence Table, Sequence ID No. 11) can be obtained by expressing cDNAin animal cells such as CHO cells, BHK cells, Namalwa, or COS-7 cells,insect cells or Escherichia coli, and purifying them using the samepurification methods as mentioned above. These may be used as antigensfor immunization. In this instance, purifying a large amount and a highlevel of mouse OBM or human OBM which are membrane-bound proteins is atask requiring a great deal of labor. On the other hand, as mentionedabove, OBM which is a membrane-bound protein and a soluble OBM (sOBM)which is obtained by deleting transmembrane domain of OBM are known tobe almost the same in their osteoclast differentiation and maturationactivities. It is possible to use mouse sOBM and human sOBM which arerelatively easily expressed and purified to a high level, as antigensfor immunization.

[0067] Mouse sOBM (Sequence Table, Sequence ID No. 16) and human sOBM(Sequence Table, Sequence ID No. 17) can be obtained by adding anucleotide sequence encoding a known signal sequence originating fromthe other secretion protein in the upstream side of the 5′ end of,respectively, mouse sOBM cDNA (Sequence Table, Sequence ID No. 18) andhuman sOBM cDNA (Sequence Table, Sequence ID No. 19), inserting theseinto an expression vector by the use of genetic engineering techniques,causing these proteins to be expressed in host cells such as variousanimal cells, insect cells, or Escherichia coli, and purifying theresultant products. The antigens for immunization thus obtained aredissolved in phosphate buffered saline (PBS), mixed with the same volumeof Freund's complete adjuvant to emulsify the solution if required, andsubcutaneously administered to animals about once a week to immunizethese animals several times. A booster injection is given when theantibody titer reaches a maximum. Exsanguination is performed 10 daysafter the booster administration. The resulting antiserum is treatedwith ammonium sulfate precipitation. IgG fraction is purified using ananion exchange chromatography or purified by protein A-or proteinG-Sepharose column chromatography after diluting the antiserum two-foldwith Binding Buffer™ (BioRad Co.), to obtain the anti-mouse oranti-human OBM/sOBM polyclonal antibody.

[0068] The monoclonal antibodies of the present invention can beobtained according to the following method. In the same manner as in thecase of the polyclonal antibodies, natural mouse OBM (native OBM),recombinant mouse or human OBM, or recombinant mouse or human sOBM canbe used as immunogens to prepare monoclonal antibodies. Hybridomas areproduced according to conventional methods by immunizing mammals withthese antigens or by immunizing lymphocytes in vitro and fusing theimmunized cells with myeloma cells. By analyzing the hybridoma culturesupernatant thus obtained by a solid phase ELISA method,antibody-producing hybridomas recognizing the highly purified antigenare selected. The resulting hybridomas are cloned and established asstable antibody-producing hybridoma clones. These hybridomas arecultured to obtain the antibodies. Small mammals such as mice or ratsare commonly used to produce hybridomas. Animals are immunized byintravenously or intraperitoneally injecting the antigen diluted to asuitable concentration using a suitable solvent such as physiologicalsalt solution. Optionally, Freund's complete adjuvant maybe usedtogether with antigen. These are usually injected 3-4 times, once a weekor every two weeks. The immunized animals are dissected three days afterfinal immunization. Splenocytes from the removed spleen are used asimmunized cells. As mouse myeloma to be fused with immunized cells,p3/×63-Ag8, p3-U1, NS-1, MPC-11, SP-2/0, FO, P3×63 Ag8.653, and S194 canbe given. A cell line such as R-210 is given as the cell of rat origin.Human antibodies are produced by immunizing human B lymphocytes in vitroand fusing the immunized cells with human myeloma cells or a cell linetransformed with EB virus. The fusion of the immunized cells and myelomacells can be carried out according to a conventional method such as themethod of Koehler and Milstein (Koehler et al.: Nature 256, 495-497(1975)). A method using electric pulse is also applicable. Immunizedlymphocytes and myeloma cells are mixed at a conventionally acceptedratio and fused in an FCS-free (fetal bovine serum-free) culture mediumwith an addition of polyethylene glycol, and cultured in anFCS-containing HAT selection medium to select fused cells (hybridomas).Next, the hybridomas which produce antibodies were selected by using aconventional antibody detection method such as an ELISA, a plaquetechnique, Ouchterlony method, or aggregation method, to establishstable hybridomas. The hybridomas established in this way can besubcultured by a conventional culture method or can be stored byfreezing as required. A hybridoma can be cultured by a conventionalmethod to collect the culture supernatant or implanted in the abdominalcavity of mammals to obtain the antibody from the ascitic fluid. Theantibody in the culture supernatant or ascitic fluid can be purified bya conventional method such as salting out, ion exchange and gelfiltration chromatography, or protein A or protein G affinitychromatography. Almost all monoclonal antibodies obtained using sOBM asan antigen can specifically recognize not only sOBM but also OBM (suchantibodies are hereinafter called anti-OBM/sOBM monoclonal antibodies).These antibodies can be used for the quantification of OBM or sOBM. Theamounts of OBM and sOBM can be quantified by labeling these antibodieswith a radioisotope or an enzyme and by applying the labeled antibodiesto a quantification system known as a radioimmunoassay (RIA) or enzymeimmunoassay (EIA). Using these quantification systems, the amount ofsOBM in a biological sample such as blood or urine can be determinedwith ease at high sensitivity. In addition, the amount of OBM binding toa tissue or surface of cells can be measured with ease at highsensitivity utilizing a binding assay using these antibodies.

[0069] When an antibody is used as a medication for humans, it isdesirable to use a human-type anti-human OBM/sOBM antibody in view ofantigenicity. The human-type anti-human OBM/sOBM antibody can beprepared according to the following methods {circle over (1)}, {circleover (2)}, or {circle over (3)}. In the method {circle over (1)}, humanlymphocytes collected from human peripheral blood or spleen areimmunized with an antigen human OBM or human sOBM in vitro in thepresence of IL-4. The resulting immunized human lymphocytes are fusedwith K6H6/B5 (ATCC CRL1823) which is a hetero hybridoma of mouse andhuman, and screened to obtain the objective antibody producinghybridoma. The antibodies produced by the resulting antibody producinghybridomas are human type anti-human OBM/sOBM monoclonal antibodies. Theantibodies neutralizing the activity of human OBM/sOBM are selected fromthese antibodies. However, in general, it is difficult to produce anantibody exhibiting high affinity to an antigen by the method ofimmunizing human lymphocytes in vitro. Therefore, in order to obtainmonoclonal antibodies with high affinity to human OBM and sOBM, it isnecessary to increase the affinity of the human-type anti-human OBM/sOBMmonoclonal antibodies obtained by the above method. This can be doneaccording to the following method. First, a random mutation isintroduced into CDR region (particularly CDR3 region) of a human-typeanti-human OBM/sOBM monoclonal antibody which neutralize OBM but have alow affinity, and make the phage to express protein. Phages which canstrongly bind to human OBM/sOBM which are selected by a phage displaymethod using plates on which human OBM/sOBM antigens are immobilized.The selected phages are grown in Escherichia coli. The amino acidsequence of the CDR which exhibits high affinity is determined from thenucleotide sequence of the DNA cloned in the phage. The thus-obtainedDNA encoding the human type anti-human OBM/sOBM monoclonal antibodies isintroduced into a commonly used expression vector for mammalian cells toproduce the human type anti-human OBM/sOBM monoclonal antibodies. Thetarget human type anti-human OBM/sOBM monoclonal antibodies exhibitinghigh affinity and capable of neutralizing the biological activity ofhuman OBM/sOBM can be selected from these monoclonal antibodies. In themethod {circle over (2)}, mouse type anti-human OBM/sOBM monoclonalantibodies are produced according to the same method as in the presentinvention using BALB/cmouse (Koehler et al.: Nature 256, 495-49, 1975),and monoclonal antibodies which can neutralize the biological activityof human OBM/sOBM and exhibiting high affinity are selected. These highaffinity mouse anti-human OBM/sOBM monoclonal antibodies can beconverted into human-type using the CDR-grafting technique (Winter andMilstein: Nature 349, 293-299, 1991) by implanting its CDR regions(CDR-1, 2 and 3) into the CDR regions of human IgG. In the method{circle over (3)}, human peripheral blood lymphocytes are implanted intoa severe combined immune deficiency (SCID) mouse. Because the implantedSCID mouse can produce human antibodies (Mosier D. E. et al.: Nature335, 256-259, 1988; Duchosal M. A. et al.: Nature 355, 258-262, 1992),lymphocytes which can produce the human monoclonal antibodies havingspecificity to human OBM/sOBM can be collected by screening SCID mouseimmunized with human OBM or sOBM. The resulting lymphocytes are fusedwith K6H6/B5 (ATCC CRL1823) which is a heterohybridoma of mouse andhuman, according to the procedure described above for the humanantibodies in the method {circle over (1)}. The resulting hybridomas arescreened to obtain hybridomas which can produce the objective humanmonoclonal antibodies. The thus-obtained hybridomas are cultured toproduce large amounts of the objective human monoclonal antibodies. Theantibodies can be purified by the above-mentioned purification method.In addition, it is possible to produce recombinant human monoclonalantibodies in large amounts by constructing a cDNA library from thehybridoma which can produce the objective human monoclonal antibodies toobtain a gene (cDNA) encoding the objective human-type monoclonalantibodies by cloning, inserting this gene into a suitable expressionvector by using genetic engineering techniques, and expressing themonoclonal antibodies in host cells such as various animal cells, insectcells, or Escherichia coli. A large amounts of purified human monoclonalantibodies can be obtained by purifying from the resulting culturesupernatant by the purification methods mentioned above.

[0070] The antibodies which can neutralize the biological activity ofOBM/sOBM can be obtained from the anti-OBM/sOBM monoclonal antibodiesproduced according to this method. The antibodies which neutralize thebiological activity of OBM/sOBM are expected to be useful as agents forthe treatment or prevention of bone metabolism abnormality because oftheir capability of blocking in vivo biological activity of OBM/sOBM,specifically the capability of preventing the induction osteoclastformation. The activity of anti-OBM/sOBM antibodies to neutralize thebiological activity of OBM or sOBM can be measured by determining theactivity to suppress osteoclast formation in the in vitro system.Specifically, the following in vitro osteoclastogenesis culture systemcan be given: {circle over (1)} a co-culture system of mouseosteoblastic stromal cell strain, ST2 cells, and mouse spleen cells inthe presence of active-form vitamin D₃ and dexamethasone, {circle over(2)} a co-culture system comprising OBM expressing monkey kidney cellstrain, COS-7, immobilizing the OBM-expressing cells with formaldehyde,and culturing mouse spleen cells on those cells in the presence ofM-CSF, and {circle over (3)}a culture system of mouse spleen cells inthe presence of recombinant sOBM and M-CSF. Theosteoclastogenesis-inhibitory activity of the anti-OBM/sOBM antibodiescan be measured by adding the anti-OBM/sOBM antibodies at variousconcentrations to these culture systems and investigating their effectson osteoclast formation. The osteoclastogenesis-inhibitory activity ofthe anti-OBM/sOBM antibodies can also be evaluated by measuring theirbone resorption-inhibitory activity utilizing experimental animals invivo. Especially, ovariectomized animal model is given as an animalmodel with progressive osteoclast formation. Theosteoclastogenesis-inhibitory activity of the anti-OBM/sOBM antibodiescan be determined by administering the anti-OBM/sOBM antibodies to suchexperimental animals and evaluating the suppression of bone resorption(a bone density increasing activity).

[0071] The thus-obtained antibodies capable of neutralizing the OBM/sOBMbiological activity are useful in pharmaceutical compositions,particularly pharmaceutical compositions to prevent or treat bonemetabolism abnormality or as antibodies for an immunological diagnosisof such diseases. The preparations comprising the antibodies of thepresent invention can be administered either orally or non-orally. Suchpreparations can be safely administered to humans or animals aspharmaceutical compositions which contain the antibodies recognizing OBMand/or sOBM as an active component. As the forms of pharmaceuticalcomposition, injection agents including intravenous drip, suppositoryagents, sublingual agents, percutaneous absorption agents, and the likeare given. Because monoclonal antibodies are macromolecule proteins,they not only readily adhere to a glass container such as a vial or asyringe, but also are easily denatured by physicochemical factors suchas heat, pH, or humidity. Therefore, the preparations should bestabilized by the addition of stabilizers, pH adjusters, bufferingagents, solubilizing agents, or detergents. As the stabilizers, aminoacids such as glycine and alanine, saccharides such as dextran 40 andmannose, and sugar alcohols such as sorbitol, mannitol, and xylytol canbe given. These stabilizers may be used either individually or incombinations of two or more. The amount of stabilizers to be added ispreferably from 0.01 to 100 times, particularly preferably from 0.1 to10 times, the amount of the antibody. The addition of these stabilizersincreases storage stability of liquid preparations or lyophilizedproducts thereof. Phosphate buffers and citrate buffers are given asexamples of the buffering agents. The buffering agents not only adjustthe pH of the liquid preparations or aqueous solutions obtained byre-dissolving the lyophilized products thereof, but also increasestability and solubility of the antibody. It is desirable to add thebuffering agent in an amount to make from 1 mM to 10 mM concentration ofthe liquid preparation or of the aqueous solution prepared from thelyophilized product. Polysolbate 20, Pulluronic F-68, and polyethyleneglycol are given as examples of the detergent. A particularly preferredexample is Polysolbate 80. These detergents may be used eitherindividually or in combinations of two or more. Macromolecule proteinssuch as an antibody is easily adhere to glass containers. Adherence tocontainers of the antibody in a liquid preparation or in an aqueoussolution prepared by re-dissolving a lyophilized product can beprevented by adding such detergents at a concentration from 0.001 to1.0%. The preparations comprising the antibodies of the presentinvention can be obtained by adding stabilizers, buffering agents, oragents which prevent adsorption. When the preparations are used asinjection agents for medication or for animals, such injection agentsshould preferably have an osmotic pressure ratio of 1 to 2. The osmoticpressure ratio can be adjusted by increasing or decreasing the amount ofsodium chloride when making the preparations. The amount of an antibodyin a preparation can be suitably adjusted depending on the disease,route of administration, and the like. A dose of a human antibody tohumans may be changed depending on the affinity of the antibody to humanOBM/sOBM, especially, on the dissociation constant (Kd value) to humanOBM/sOBM. The higher the affinity (or the smaller the Kd value), theless the dose to be administered to humans to obtain a certain medicinaleffect. Because a human-type antibody has a long half-life in blood ofabout 20 days, it is sufficient to administer it to humans at a dose ofabout 0.1-100 mg/kg once or more in a 1-30 day period.

BRIEF DESCRIPTION OF THE DRAWINGS

[0072]FIG. 1 shows the result of SDS-PAGE of mouse OBM protein of thepresent invention obtained in Example 3.

[0073] Explanation of Symbols

[0074] (A): Lane 1: Molecular weight markers

[0075] Lane 2: A partially purified sample (Gly-HCl (pH 2.0) elutionfraction) obtained from ST2 cells cultured in the presence ofactive-form vitamin D₃ and dexamethasone.

[0076] Lane 3: A partially purified sample (Gly-HCl (pH 2.0) elutionfraction) obtained from ST2 cells cultured in the absence of active-formvitamin D₃ and dexamethasone.

[0077] (B): Lane 1: Molecular weight markers

[0078] Lane 2: Mouse OBM protein of the present invention afterpurification by reverse phase high performance liquid chromatography(Example 3)

[0079]FIG. 2 shows the result of the binding assay of ¹²⁵I labeled OCIFto osteoblastic stromal cells, ST2, in Example 4.

[0080]FIG. 3 shows the osteoclast formation capability of osteoblasticstromal cells ST2 from different generations in Example 5(1).

[0081] Explanation of Symbols

[0082] 1: Ability of ST2 cells from about a 10^(th) subculture tosupport osteoclast formation.

[0083] 2: Ability of ST2 cells from about a 40^(th) subculture tosupport osteoclast formation.

[0084]FIG. 4 shows change with the passage of time in expression of theprotein of the present invention on the cell membrane of osteoblasticstromal cells cultured in the presence of active-form vitamin D₃ anddexamethasone in Example 5(2).

[0085]FIG. 5 shows change with the passage of time in osteoclastformation in the co-culture system of Example 5(2).

[0086]FIG. 6 shows the inhibitory effect on osteoclast formation whentreated with OCIF for different culture periods during the co-cultureperiod in Example 5(3).

[0087]FIG. 7 shows the results of a crosslinking test of ¹²⁵I-labeledOCIF with the protein of the present invention in Example 6.

[0088] Explanation of Symbols

[0089] Lane 1: ¹²⁵I-labeled OCIF-CDD1

[0090] Lane 2: ¹²⁵I-labeled OCIF-CDD1 crosslinked with ST2 cells

[0091] Lane 3: ¹²⁵I-labeled OCIF-CDD1 crosslinked in the presence of400-fold excess of unlabeled OCIF

[0092]FIG. 8 shows the result of SDS-PAGE in Example 9.

[0093] Explanation of Symbols

[0094] Lane 1: Proteins of pOBM291-transfected COS-7 cellsimmonoprecipitated in the absence of OCIF

[0095] Lane 2: Proteins of pOBM291-transfected COS-7 cellsimmunoprecipitated in the presence of OCIF

[0096]FIG. 9 shows the results of analysis of binding capability of¹²⁵I-labeled OCIF to COS-7 cells transfected with pOBM291 in Example 10.

[0097] Explanation of Symbols

[0098] Lanes 1 and 2: The amount of the ¹²⁵I-labeled OCIF binding toCOS-7 cells transfected with pOBM291

[0099] Lanes 3 and 4: The amount of the ¹²⁵I-labeled OCIF binding toCOS-7 cells transfected with pOBM291 in the presence of 400-fold excessof unlabeled OCIF

[0100]FIG. 10 shows the result of a crosslinking test using OCIF labeledwith ¹²⁵I in Example 11.

[0101] Explanation of Symbols

[0102] Lane 1: ¹²⁵I-labeled OCIF

[0103] Lane 2: ¹²⁵I-labeled OCIF crosslinked with COS-7 cellstransfected with pOBM291

[0104] Lane 3: ¹²¹I-labeled OCIF crosslinked with COS-7 cellstransfected with pOBM291 in the presence of 400-fold excess of unlabeledOCIF

[0105]FIG. 11 shows the result of a Northern Blot in Example 12.

[0106] Explanation of Symbols

[0107] Lane 1: RNA originating from ST2 cells cultured without additionof Vitamin D and dexamethasone

[0108] Lane 2: RNA originating from ST2 cells cultured with the additionof Vitamin D and dexamethasone

[0109]FIG. 12 shows the OCIF-binding capability of the proteins in theconditioned medium at various OCIF concentrations in Example 13(2).

[0110] Explanation of Symbols

[0111] ◯: pCEP4

[0112] : pCEP sOBM

[0113]FIG. 13 shows the OCIF-binding capability of the protein in theconditioned medium at various proportions of the conditioned medium inExample 13(2).

[0114] Explanation of Symbols

[0115] ◯: PCEP4

[0116] : PCEP sOBM

[0117]FIG. 14 shows the result of SDS-PAGE of a fusion proteinconsisting of thioredoxin and mouse OBM expressed in Escherichia coli inExample 14(2).

[0118] Explanation of Symbols

[0119] Lane 1: Molecular weight markers

[0120] Lane 2: Soluble protein fractions originating from GI724/pTrxFus

[0121] Lane 3: Soluble protein fractions originating fromGI724/pTrxOBM25

[0122]FIG. 15 shows the OCIF-binding capability at various proportionsof soluble protein fractions in Example 14(3).

[0123] Explanation of Symbols

[0124] □: GI724/pTrxFus

[0125] ◯: GI724/pTrxOBM25

[0126]FIG. 16 shows the OCIF-binding capability of soluble proteinfractions (1%) at various concentrations of OCIF in Example 14(3).

[0127] Explanation of Symbols

[0128] □: GI724/pTrxFus

[0129] ◯: GI724/pTrxOBM25

[0130]FIG. 17 shows the results of inhibition of specific binding toOCIF of mouse OBM obtained by expression of mouse OBM cDNA of thepresent invention and purification or natural OCIF-binding protein by arabbit anti-mouse OBM antibody.

[0131] Explanation of Symbols

[0132] 1: Purified OBM prepared by expression of the cDNA in thepresence of the antibody, OBM+¹²⁵I-OCIF

[0133] 2: The natural protein in the presence of the antibody+¹²⁵I-OCIF

[0134] 3: Mouse OBM prepared by expression of the cDNA in the absence ofthe antibody, mouse OBM+¹²⁵I-OCIF

[0135] 4: The natural protein in the absence of the antibody+¹²⁵I-OCIF

[0136] 5: 3+unlabeled OCIF (400-fold more than ¹²⁵I-OCIF)

[0137] 6: 4+unlabeled OCIF (400-fold more than ¹²⁵I-OCIF)

[0138]FIG. 18 shows the result of SDS-PAGE of human OBM proteinexpressed by the cDNA of the present invention.

[0139] Explanation of Symbols

[0140] Lane 1: Molecular weight markers

[0141] Lane 2: Proteins of COS-7 cells transfected with phOBM (anexpression vector containing a cDNA of the present invention),immunoprecipitated with a rabbit anti-OCIF polyclonal antibody in theabsence of OCIF

[0142] Lane 3: Proteins of COS-7 cells transfected with phOBM (anexpression vector containing a cDNA of the present invention),immunoprecipitated with a rabbit anti-OCIF polyclonal antibody in thepresence of OCIF

[0143]FIG. 19 shows the result of analysis of binding of OCIF to COS-7cells transfected with phOBM, an expression vector containing a cDNA ofthe present invention.

[0144] Explanation of Symbols

[0145] Lane 1: COS-7 cells transfected with phOBM and the addition of¹²⁵I-OCIF.

[0146] Lane 2: COS-7 cells transfected with phOBM and the addition of¹²⁵I-OCIF, in the presence of a 400-fold more unlabeled OCIF

[0147]FIG. 20 shows the result of crosslinking of human OBM, which is aprotein encoded by a cDNA of the present invention, with ¹²⁵I-OCIF(monomer-type).

[0148] Explanation of Symbols

[0149] Lane 1: ¹²⁵I-OCIF

[0150] Lane 2: The crosslinked products of ¹²⁵I-OCIF with the proteinson the membrane of COS-7 cells transfected with phOBM.

[0151] Lane 3: The crosslinked products of ¹²⁵-OCIF with the proteins onthe membrane of COS-7 cells transfected with phOBM, in the presence of a400-fold more unlabeled OCIF.

[0152]FIG. 21 shows the OCIF-binding capability of the protein(secreted-form hOBM) in the conditioned medium at various OCIFconcentrations in Example 23(2).

[0153] Explanation of Symbols

[0154] ◯: Conditioned medium of 293-EBNA cells transfected with pCEP4,which does not contain cDNA encoding secreted-form human OBM

[0155] : Conditioned medium of 293-EBNA cells transfected withpCEPshOBM, which contains cDNA encoding secreted-form human OBM

[0156]FIG. 22 shows the OCIF-binding capability of the protein(secreted-form human OBM) in the conditioned medium at a specific OCIFconcentration while changing the amount of conditioned medium added inExample 23(2).

[0157] Explanation of Symbols

[0158] ◯: Conditioned medium of 293-EBNA cells transfected with pCEP4,which does not contain cDNA encoding secreted-form human OBM

[0159] : Conditioned medium of 293-EBNA cells transfected withpCEPshOBM, which contains cDNA encoding secreted-form human OBM

[0160]FIG. 23 shows the result of SDS-PAGE of a fusion proteinconsisting of thioredoxin and human OBM expressed in Escherichia coli.

[0161] Explanation of Symbols

[0162] Lane 1: Molecular weight markers

[0163] Lane 2: Soluble protein fractions originating from Escherichiacoli GI724/pTrxFus

[0164] Lane 3: Soluble protein fractions originating from Escherichiacoli GI724/pTrxhOBM

[0165]FIG. 24 shows the OCIF-binding capability of the fusion proteinconsisting of thioredoxin and human OBM to OCIF, when the amount of thesoluble protein fraction originating from Escherichia coli including thefusion protein added was varied in Example 24(3).

[0166] Explanation of Symbols

[0167] ◯: Soluble protein fractions originating from Escherichia coliGI724/pTrxFus

[0168] : Soluble protein fractions originating from Escherichia coliGI724/pTrxshOBM

[0169]FIG. 25 shows the OCIF-binding capability of the fusion protein ofthioredoxin and human OBM in soluble protein fractions originating fromEscherichia coli to OCIF in various concentrations in Example 24(3).

[0170] Explanation of Symbols

[0171] ◯: Soluble protein fractions originating from Escherichia coliGI724/pTrxFus

[0172] : Soluble protein fractions originating from Escherichia coliGI724/pTrxshOBM

[0173]FIG. 26 shows the result of quantifying human OBM and human sOBMby the sandwich ELISA method using the rabbit anti-human OBM/sOBMpolyclonal antibody of the present invention.

[0174] Explanation of Symbols

[0175] □: Human OBM

[0176] : Human sOBM

[0177]FIG. 27 shows the result of quantifying human OBM and human sOBMby the sandwich ELISA method using the anti-human OBM/sOBM monoclonalantibodies of the present invention.

[0178] Explanation of Symbols

[0179] □: Human OBM

[0180] : Human sOBM

[0181]FIG. 28 shows the result of quantifying mouse OBM and sOBM by thesandwich ELISA method using the anti-human OBM/sOBM monoclonalantibodies of the present invention which cross react mouse OBM andsOBM.

[0182] Explanation of Symbols

[0183] □: Mouse OBM

[0184] : Mouse sOBM

[0185]FIG. 29 shows the activity of the fusion protein consisting ofthioredoxin and mouse OBM to stimulate human osteoclast-like cellformation.

[0186]FIG. 30 shows the suppression of the anti-OBM/sOBM antibody of thebone resorption activity stimulated by vitamin D₃.

[0187]FIG. 31 shows the suppression of the anti-OBM/sOBM antibody of thebone resorption activity stimulated by prostaglandin E₂ (PGE₂).

[0188]FIG. 32 shows the suppression by the anti-OBM/sOBM antibody of thebone-resorbing activity stimulated by parathyroid hormone (PTH).

[0189]FIG. 33 shows the suppression by the anti-OBM/sOBM antibody of thebone-resorbing activity stimulated by interleukin 1 α (1L-1)

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLES

[0190] The present invention will be described in more detail by way ofexamples which are given for the purpose of illustration of theinvention and are not limiting thereof in any way of the remainder ofthe disclosure.

Example 1

[0191] Preparation of the Protein of the Present Invention

[0192] (1) Large-Scale Cultivation of ST2 Cells

[0193] Mouse osteoblastic stromal cell line ST2 (RIKEN CELL BANKRCB0224) was cultured using α-MEM containing 10% fetal bovine serum. ST2cells cultured to confluence in a 225 cm² T flask for adherent-cellculture were treated with trypsin and harvested from the T flask. Afterwashing, the cells were transferred to five 225 cm² T flasks. After theaddition of 60 ml of α-MEM containing 10⁻⁸ M active-form vitamin D₃(Calcitriol), 10⁻⁷ M dexamethasone, and 10% fetal bovine serum, cells ineach flask were cultured for 7-10 days in a CO₂ incubator. The culturedST2 cells were harvested using a cell scraper and stored at −80° C.until use.

[0194] (2) Preparation of Membrane Fraction and Solubilization ofMembrane-Bound Proteins

[0195] To the ST2 cells (volume, about 12 ml) described in Example 1(1),cultured using eighty 225 cm² T flasks, was added three times the volume(36 ml) of 10 mM Tris-HCl buffer (pH 7.2) containing protease inhibitors(2 mM APMSFP, 2 mM EDTA, 2 mM o-phenanthroline, 1 mM leupeptin, 1 μg/mlpepstatin A, and 100 unit/ml aprotinin). After vigorously agitating for30 seconds using a voltex mixer, the cells were allowed to stand for 10minutes on ice. The cells were homogenized using a homogenizer (DOUNCETISSUE GRINDER, A syringe, WHEATON SCIENTIFIC Co.). The same volume (48ml) of 10 mM Tris-HCl buffer (pH 7.2) containing the above-mentionedprotease inhibitors, 0.5 M sucrose, 0.1 M potassium chloride, 10 mMmagnesium chloride, and 2 mM calcium chloride was added to thehomogenized cells. After stirring, the mixture was centrifuged at 600×gfor 10 minutes at 4° C., thereby separating nuclei and non-homogenizedcells as precipitate. The supernatant obtained by the centrifuge wascentrifuged at 150,000×g for 90 minutes at 4° C., to obtain membranefraction of the ST2 cells as precipitate. Eight ml of 10 mM Tris-HClbuffer (pH 7.2) containing the above-mentioned protease inhibitors, 150mM sodium chloride, and 0.1 M sucrose was added to this membranefraction. After the addition of 200 μl of 20% CHAPS(3-[(3-cholamidopropyl)-dimethylamonio]-1-propanesulfonate, Sigma Co.),the mixture was stirred for 2 hours at 4° C. The mixture was thencentrifuged at 150,000×g for 60 minutes at 4° C., to obtain supernatantas solubilized membrane fraction.

Example 2

[0196] Purification of the Protein of the Present Invention

[0197] (1) Preparation of OCIF-Immobilized Affinity Column

[0198] After replacing iso-propanol in a HiTrap NHS-activated column (1ml, manufactured by Pharmacia Co.) with 1 mM hydrochloric acid, 1 ml of0.2 M NaHCO₃/0.5 M NaCl solution (pH 8.3) containing 13.0 mg ofrecombinant OCIF prepared by the method of WO 96/26217 was added to thecolumn using a syringe (5 ml, manufactured by Terumo Corp.), to effectcoupling reaction at room temperature for 30 minutes. The column was fedwith 3 ml of 0.5 M ethanolamine/0.5 M NaCl (pH 8.3) and 3 ml of 0.1 Macetic acid/0.5 M NaCl (pH 4.0) three times in turn to inactivate excessactive groups, then the solution was replaced with 0.5 Methanolamine/0.5 M NaCl (pH 8.3). After allowing to stand at roomtemperature for 1 hour, the column was washed twice alternately with 0.5M ethanolamine/0.5 M NaCl (pH 8.3) and 0.1 M acetic acid/0.5M NaCl (pH4.0), and the solution was then replaced with 50 mM Tris/1 M NaCl/0.1%CHAPS buffer (pH 7.5).

[0199] (2) Purification of the Protein of the Present Invention UsingOCIF-Immobilized Affinity Column

[0200] The purification of the OCIF-binding protein was carried out at4° C., unless otherwise indicated. The above-mentioned OCIF-immobilizedaffinity column was equilibrated with 10 mM Tris-hydrochloride buffer(pH 7.2) to which the protease inhibitors described in Example 1(2),0.15 M sodium chloride, and 0.5% CHAPS were added. About 8 ml of thesolubilizedmembrane fraction described in Example 1(2) was applied tothe column at a flow rate of 0.01 ml/minute. Then, the column was washedwith 10 mM Tris-hydrochloride buffer (pH 7.2) to which theabove-mentioned protease inhibitors, 0.15 M sodium chloride, and 0.5%CHAPS was added, for 100 minutes at a flow rate of 0.5 ml/minute. Next,the proteins adsorbed to the column was eluted with 0.1 Mglycine-hydrochloride buffer (pH 3.3) containing the proteaseinhibitors, 0.2 M sodium chloride, and 0.5% CHAPS for 50 minutes at aflow rate of 0.1 ml/minute. In the same manner, the proteins adsorbed tothe column was eluted with 0.1 M sodium citrate buffer (pH 2.0)containing the protease inhibitors, 0.2 M sodium chloride, and 0.5%CHAPS for 50 minutes at a flow rate of 0.1 ml/minute. The eluate wascollected in 0.5 ml fractions. Each fraction was immediately neutralizedby the addition of 2M Tris solution. The fractions derived from theelution with these buffers (each fraction consisting of 1.0-5.0 ml ofeluate) were concentrated to 50-100 μl using Centricon-10 (manufacturedby Amicon of U.S.A.). OCIF was added to a portion of each concentratedfraction and immunoprecipitated with anti-OCIF polyclonal antibody. Theprecipitated fractions were treated with SDS and subjected to SDS-PAGE.Fractions (Fr. No. 3-10) in which the band of the protein with specificbinding ability to OCIF appeared were regarded as the protein fractionsof the present invention.

[0201] (3) Purification of the Protein of the Present Invention by GelFiltration

[0202] The concentrated OCIF-binding protein (the fractions obtain bythe elution with 0.1 M glycine-hydrochloride buffer (pH 3.3) and 0.1 Msodium citrate buffer (pH 2.0)) prepared in Example 2(2) was applied toa Superose 12 HR10/30 column (1.0×30 cm, manufactured by Pharmacia Co.)which was equilibrated with 10 mM Tris-HCl, 0.5 M NaCl, 0.5% CHAPS(pH7.0) and developed with the equilibration buffer as a mobile phase ata flow rate of 0.5 ml/min, and each 0.5 ml fraction was collected. Thefractions containing the protein of the present invention (Fr. Nos.27-32) were identified according to the same method as described above.Each of the fractions was concentrated using Centricon-10 (a product ofAmicon).

[0203] (4) Purification by Reverse Phase High Performance LiquidChromatography

[0204] The above-mentioned OCIF-binding protein purified by the gelfiltration was applied to a C₄ column (2.1×250 mm, Vydac, USA) which wasequilibrated with 0.1% trifluoroacetic acid (TFA) and 30% acetonitrile.The proteins bound to the column were eluted with linear gradients ofacetonitrile from 30% to 55% for the first 50 minutes and from 55% to80% during the next 10 minutes at a flow rate of 0.2 ml/min. Peaks ofeluted proteins were detected by measuring optical density at 215 nm.Proteins in the different peaks were analyzed to identify the fractionscontaining the protein of the present invention, and a highly purifiedprotein of the present invention was obtained.

Example 3

[0205] SDS-PAGE of the Purified Protein of the Present Invention

[0206] The solubilized membrane fraction prepared from ST2 cells whichwere cultured in the presence or absence of active-form vitamin D₃ wassubjected to purification with the OCIF-immobilized affinity column. Thepurified preparations were subjected to SDS-PAGE. As shown in FIG. 1(A),a major protein band with MW of about 30,000-40,000 was detected only inthe purified preparation from ST2 cells which was cultured in thepresence of the active-form vitamin D₃, proving that the protein whichspecifically binds to OCIF (i.e. the protein of the present invention)can be selectively purified by the OCIF-immobilized affinity column.However, bands of several proteins (other than the protein of thepresent invention) which non-specifically bind to carriers or spacers ofthe OCIF-immobilized column were detected in both of the purifiedpreparations. The proteins other than the protein of the presentinvention were removed according to the above-described method by gelfiltration and C4 reverse phase chromatography. SDS-PAGE of the obtainedhighly purified protein of the present invention is shown in FIG. 1(B).The highly purified protein of the present invention was found to beelectrophoretically homogeneous and had a molecular weight of about30,000-40,000.

Example 4

[0207] Binding Test of OCIF to Osteoblasts

[0208] (1) Preparation of ¹²⁵I-Labeled OCIF

[0209] OCIF was labeled with ¹²⁵I by the Iodogen method. Specifically,20 μl of 2.5 mg/ml Iodogen-chloroform solution was transferred to a 1.5ml Eppendorf tube and chloroform was evaporated off at 40° C., to obtaina tube coated with Iodogen. The tube was washed three times with 400 μlof 0.5 M sodium phosphate buffer (Na-Pi, pH 7.0). Five μl of 0.5 M Na-Pi(pH 7.0) was added to the tube. Immediately after the addition of 1.3 μl(18.5 MBq) of Na-¹²⁵I solution (NEZ-033H20, manufactured by AmershamCo.); 10 μl of 1 mg/ml rOCIF solution (monomer type or dimer type) wasadded to the tube. After mixing with a voltex mixer, the mixture wasallowed to stand at room temperature for 30 seconds. The solution wastransferred to a tube containing 80 μl of a solution of 10 mg/mlpotassium iodide in 0.5 M Na-Pi (pH 7.0) and 5 μl of a phosphatebuffered saline containing 5% bovine serum albumin, and stirred. Themixture was applied to a spin column (1 ml, G-25 fine, manufactured byPharmacia Co.) which was equilibrated with phosphate buffered salinecontaining 0.25% bovine serum albumin and the column was centrifuged for5 minutes at 2,000 rpm. Four hundred μl of a phosphate buffered salinecontaining 0.25% bovine serum albumin was added to the fraction elutedfrom the column and the mixture was stirred. A 2 μl of the aliquot wasremoved to measure the radioactivity using a gamma counter. Theradiochemical purity of the ¹²⁵I-labled OCIF was determined by measuringthe radioactivity precipitated with 10% TCA. The biological activity ofthe ¹²⁵I-labeled OCIF was measured according to the method described inWO 96/26217. The concentration of the ¹²⁵I-labeled OCIF was measured bythe ELISA according to the following procedure.

[0210] (2) Measurement of the Concentration of ¹²⁵I-Labeled OCIF byELISA

[0211] One hundred μl of 50 mM NaHCO₃ (pH 9.6) in which the anti-OCIFrabbit polyclonal antibody described in WO 96/26217 was dissolved to aconcentration of 2 μg/ml was added to each well of a 96-wellimmuno-plate (MaxiSorp™, a product of Nunc Co.). The plate-was allowedto stand overnight at 4° C. After removing the solution by suction, 300μl of Block Ace™ (Snow Brand Milk Products Co., Ltd.)/phosphate bufferedsaline (25/75) solution was added to each well. The plate was thenallowed to stand for two hours at room temperature. After removing thesolution by suction, the wells were washed three times with phosphatebuffered saline containing 0.01% Polysorbate 80 (P-PBS). Next, 300 μl ofBlock Ace™/phosphate buffered saline (25/75) solution to which¹²⁵I-labeled OCIF or the standard OCIF preparation was mixed, was addedto each well. The plate was then allowed to stand for two hours at roomtemperature. After removing the solution by suction, each well waswashed six times with 200 μl of P-PBS.

[0212] One hundred μl of Block Ace™ (Snow Brand Milk Products Co.,Ltd.)/phosphate buffered saline (25/75) solution containing peroxidaselabeled rabbit anti-OCIF polyclonal antibody was added to each well. Theplate was allowed to stand for two hour sat room temperature. Afterremoving the solution by suction, the wells were washed six times with200 μl P-PBS. Then, 100 μl of TMB solution (TMB Soluble Reagent, HighSensitivity, Scytek Co.) was added to each well. After incubating atroom temperature for 2-3 minutes, 100 μl of stopping solution (StoppingReagent, Scytek Co.) was added to each well. Absorbance of each well wasmeasured at 490 nm using a microplate reader. The concentration of¹²⁵I-labeled OCIF was determined from a calibration curve prepared usingthe standard preparation of OCIF.

[0213] (3) Binding Test of OCIF to Osteoblasts or Spleen Cells

[0214] Mouse osteoblastic stromal cell line ST2 or spleen cells weresuspended in α-MEM containing 10% fetal bovine serum (FBS), either withor without 10⁻⁸ M active-form vitamin D₃ (Calcitriol) and 10⁻⁷ Mdexamethasone, to a concentration of 4×10⁴ cells/ml (ST2 cells) or 2×10⁶cells/ml (spleen cells), respectively. Each cell suspension wasinnoculated into a 24-well micro plate. The cells were cultured for 4days in a CO₂ incubator. After washing the cells with α-MEM, 200 μl ofmedium for the binding test (α-MEM to which 0.2% bovine serum albumin,20 mM Hepes buffer, and 0.2% NaN₃ were supplemented), containing 20ng/ml of above-described ¹²⁵I-labeled OCIF (monomer form or dimer form),was added to each well. To the wells for the measurement of non-specificbinding, 200 μl of the medium for the binding test containing 8 μg/ml ofrOCIF (400 times concentration) in addition to 20 ng/ml of ¹²⁵I-labeledOCIF was added. The cells were cultured for one hour in a CO₂ incubatorand washed 3 times with 1 ml of a phosphate buffered saline. In thisprocedure, spleen cells were washed by centrifuging the 24-well plate ineach washing step, because the spleen cells were non-adherent. Afterwashing, 500 μl of 0.1 N NaOH solution was added to each well and theplate was allowed to stand for 10 minutes at room temperature todissolve the cells. The amount of RI in each well was measured by agamma counter.

[0215] As shown in FIG. 2, ¹²⁵I-labeled OCIF did not bind to thecultured spleen cells, but specifically bound only to the osteoblasticstromal cells which were cultured in the presence of active-form vitaminD₃. The results indicated that the protein of the present invention is amembrane bound protein induced by active-form vitamin D₃ anddexamethasone on osteoblastic stromal cells.

Example 5

[0216] Biological Activity of the Protein of the Present Invention

[0217] (1) Osteoclasts-Formation Supported by Osteoblastic Stromal Cells

[0218] The osteoclasts formation-supporting capability of osteoblasticstromal cells was evaluated by measuring tartaric acid resistant acidphosphatase activity (TRAP activity) of the formed osteoclasts.Specifically, spleen cells (2×10⁵ cells/100 μl/well) from a ddy mouse(8-12 weeks old) and mouse osteoblastic stromal cells ST2 (5×10³cells/100 μl/well) were suspended in α-MEM to which 10⁻⁸ M active-formvitamin D₃, 10⁻⁷ M dexamethasone, and 10% fetal bovine serum were added.The cells were innoculated into 96-well plates and cultured for one weekin a CO₂ incubator. After washing each well with phosphate bufferedsaline, 100 μl of ethanol/acetone (1:1) was added to each well, and thecells were immobilized at room temperature for one minute. Afterimmobilization, 100 μl of 50 mM citrate buffer (pH 4.5) containing 5.5mM p-nitrophenol phosphate and 10 mM sodium tartarate was added to eachwell. After 15 minutes of reaction at room temperature, 0.1 N NaOHsolution was added to each well and absorbance at 405 nm was measuredusing a microplate reader. The results of osteoclasts-formation by ST2cells with a passage number of about 10 or 40 after purchasing the cellsfrom RIKEN CELL BANK are shown in FIG. 3. The results indicate that theST2 cells with a higher passage number exhibit more potent ability tosupport osteoclasts-formation.

[0219] (2) Time Course Change of Expression of the Protein of thePresent Invention on Membrane of Osteoblastic Stromal Cells in a CultureSystem Which Include Active-Form Vitamin D₃ and Dexamethasone and TimeCourse Change of Osteoclasts-Formation in the Co-Culture System

[0220] In the same manner as in Example 4(3), osteoblastic stromal cellST2 was cultured for 7 days in the presence of active-form vitamin D₃and dexamethasone. The OCIF-binding test was carried out using¹²⁵I-labeled OCIF (monomer type) as described in the experiment inExample 4(1). Non-specific binding was measured by competing ¹²⁵I-OCIFbinding to ST2 cells with 400-fold concentration of unlabeled OCIF. As aresult, it was confirmed that the amount of specific binding of¹²⁵I-labeled OCIF increase in accordance with increase in culture periodin the presence of active-form vitamin D₃ and dexamethasone.Specifically, as shown in FIGS. 4 and 5, the protein of the presentinvention was expressed on the surface of ST2 cells by active-formvitamin D₃ in accordance with increase in culture period and theexpression reached a maximum on the fourth day of culture. On the otherhand, osteoclast-like cells are formed by coculturing mouse spleen cellsand ST2 cells in the presence of active-form vitamin D₃. TRAP (a markerenzyme of osteoclasts) -positive mononuclear pre-osteoclast-like cellsare formed on the third or fourth day of the culture. Moredifferentiated and mature TRAP-positive multinuclear cells are formed onthe fifth to sixth day of the culture. A good correlation betweentime-course of the expression of the protein of the present inventionand osteoclasts-formation was thus demonstrated.

[0221] (3) Inhibition of Osteoclasts Formation by OCIF Treatment forDifferent Period During the Co-Culture

[0222] To make it clear that the protein of the present invention is afactor involved in the osteoclasts-formation, the cells were treatedwith 100 mg/ml OCIF for different culture periods during the six dayco-culture period described in the above-mentioned Example 5(2) (twoconsequtive days in the six-day period, except for the 5th day for whicha one-day period was applied). As a result, as shown in FIG. 6, OCIFtreatment at 48-96 hours after start of the culture at which expressionof the protein of the present invention on ST2 cells is maximal wasfound to be most effective for inhibiting formation of osteoclasts.Specifically, it was confirmed that OCIF controls osteoclast formationby binding to ST2 cells via the protein of the present invention.

[0223] Based on the results of the above experiments, the protein of thepresent invention was confirmed to be induced on cell membrane ofosteoblastic stromal cells by active-form vitamin D₃ and dexamethasoneand to exhibit a biological activity to support or acceleratedifferentiation or maturation of osteoclasts.

Example 6

[0224] Crosslinking test for ¹²⁵I-Labeled OCIF and the Protein of thePresent Invention

[0225] To identify the protein of the present invention more clearly,the protein of the present invention was crosslinked with ¹²⁵I-labeledOCIF. Mouse osteoblastic stromal cell line ST2 was cultured for fourdays in the presence or absence of active-form vitamin D₃ anddexamethasone in the same manner as described in Example 4(3). Afterwashing the cells with 1 ml of phosphate buffered saline, 200 μl ofmedium for binding test (α-MEM to which 0.2% bovine serum albumin, 20 mMHepes buffer, 0.2% NaN₃, and 100 μg/ml heparin were added) ,containing25 ng/ml of ¹²⁵-labeled OCIF (monomer type) or 40 ng/ml of ¹²⁵I-labeledOCIF-CDD1 which was obtained by expressing the protein of Sequence IDNo. 76 (WO 96/26217) in animal cells, was added. The above-mentionedculture medium for the binding test was further supplemented with400-fold concentration of OCIF and was added to the other wells toassess non-specific binding. After culturing for one hour in a CO₂incubator, each well was washed three times with 1 ml of phosphatebuffered saline containing 100 μg/ml heparin. Five hundred μl ofphosphate buffered saline containing 100 μg/ml crosslinking agent, DSS(Disuccinimidyl suberate, Pierce Co.), was added to each well and theplate was kept for 10 minutes at 0° C. The wells were washed twice with1 ml of phosphate buffered saline at 0° C. One hundred μl of 20 mM Hepesbuffer containing 1% Triton X-100, 10 μM pepstatin, 10 μM leupeptin, 2mM PMSF (phenylmethylsulfonyl fluoride), 10 μM antipain, and 2 mM EDTA,was then added to each well. The plate was allowed to stand for 30minutes at room temperature to dissolve the cells. Fifteen μl of thesesamples were treated with SDS under non-reducing conditions according toconventional method and subjected to SDS-polyacrylamide gelelectrophoresis(4-20% polyacrylamide gradient, manufactured by DaiichiChemical Co., Ltd.). After electrophoresis, the gels were dried andexposed to BioMax MS film (manufactured by Kodak) for 24 hours at −80°C. using BioMax MS intensifying screens (manufactured by Kodak). Afterexposure, the film was developed by conventional method. A band ofcrosslinking product with a molecular weight of 90,000-110,000 wasdetected when the ¹²⁵I-labeled OCIF (monomer type, 60 kDa) was used.When the ¹²⁵I-labeled OCIF-CDD1 (31 kDa) was used, a band ofcrosslinking product of about 70-80 kDa (average, 78 kDa) was detectedas shown in FIG. 7.

Example 7

[0226] Analysis of the Protein of the Present Invention Expressed on STCells by Scatchard Plot

[0227] The above-mentioned ¹²⁵I-labeled OCIF (monomer type) was added toa concentration of 1,000 pM to the culture medium for binding test(α-MEM containing 0.2% bovine serum albumin, 20 mM Hepes buffer, and0.2% NaN₃) and the culture medium was serially diluted at a rate to ½with the culture medium not containing ¹²⁵I-labeled OCIF. Solutions formeasuring non-specific binding were prepared by further adding 400-foldconcentration of monomer-form OCIF to these solutions. Two hundred μl ofthe prepared solutions were added to the above-mentioned wells with ST2cells cultured for 4 days (passage number, about 10) in the presence of10⁻⁸ M active-form vitamin D₃ (Calcitriol) and 10⁻⁷ M dexamethasone, toassess binding of ¹²⁵I-labeled OCIF in the same method as described inExample 4(3). The results were subjected to Scatchard Plot analysis todetermine the dissociation constant of OCIF and OCIF-binding protein andthe number (site) of OCIF-binding protein per a ST2 cell. As a result,the dissociation constant of OCIF and the protein of the presentinvention was found to be 280 pM, and the number of the site ofOCIF-binding protein per a ST2 cell was approximately 33,000/cell. Basedon the finding in Example 5(1) that osteoclasts-formation supported bythe ST2 cells with passage number about 40 was more extensive than thatwith passage number about 10, the number (the site) of the protein ofthe present invention expressed on the ST2 cell with a passage numberabout 40 was assessed. The number (site) was 58,000/cell and was clearlylarger than the ST2 cells with passage number about 10, indicating thatthe amount of the protein of the present invention expressed on ST2cells is related to their potency to support osteoclasts-formation. Theresults indicated that the protein of the present invention is a factorthat supports or induces differentiation or maturation of osteoclasts.

Example 8

[0228] Cloning of OBMcDNA

[0229] (1) Extraction of RNA from Mouse ST2 Cells

[0230] Mouse osteoblastic stromal cell line ST2 (RIKEN CELL BANK,RCB0224) was cultured in α-MEM (Gibco BRL Co.) containing 10% fetalbovine serum. ST2 cells cultured to confluent in a 225 cm² T-flask foradherent cells were treated with trypsin to harvest the cells from theT-flask. The cells were washed and transferred to five 225 cm² T-flasks.Sixty ml of α-MEM containing 10⁻⁸ M active-form vitamin D₃ (Calcitriol,Wako Pure Chemicals Co., Ltd.), 10⁻⁷ M dexamethasone, and 10% fetalbovine serum was added to each flask and the cells were cultured for 5days in a CO₂ incubator. Total RNA was extracted from the cultured ST2cells using ISOGEN (Wako Pure Chemicals Co., Ltd.). Poly A⁺RNA wasprepared from about 600 μg of the total RNA using an Oligo(dT)-cellulose column (5′-3′ Prime Co.). About 8 μg of Poly A⁺RNA wasobtained.

[0231] (2) Construction of Expression Library

[0232] Double-stranded cDNA was synthesized from 2 μg of polyA⁺RNAobtained in Example 8(1) using a Great Lengths cDNA Synthesis kit(Clontech Co.) according to the instruction in the manual. Specifically,2 μg of poly A⁺RNA and Oligo (dT)₂₅(dN) primer were mixed and distilledwater was added to the mixture to make the final volume to 6.25 μl.After incubation for about 3 minutes at 70° C., the mixture was cooledon ice for 2 minutes. To the mixture were added 2.2 μl of distilledwater, 2.5 μl of 5× First-strand buffer, 0.25 μl of 100 mM DTT(dithiothreitol) 0.5 μl of PRIME RNase inhibitor (1U/ml) (5′-3′ PrimeCo.), 0.5 μl of [α-³²P]dCTP (Amersham Co., 3000 Ci/mmol) diluted 5-foldwith distilled water to make 2 μCi/μl, 0.65 μl of dNTP (20 mM each), and1.25 μl (250 unit) of MMLV (RNaseH⁻) reverse transcriptase. The mixturewas incubated for 90 minutes at 42° C., followed by the further additionof 62.25 μl of distilled water, 20 μl of 5× second-strand buffer, 0.75μl of dNTP (20 mM each), and 5 μl of Second-strand enzyme cocktail. Theresulting mixture was maintained at 16° C. for two hours Then, 7.5 unitsof T4DNA polymerase was added to this reaction mixture. After incubationat 16° C. for 30 minutes, the reaction was terminated by the addition of5 μl of 0.2M EDTA. After a phenol-chloroform treatment, the product wasprecipitated with ethanol. An EcoRI-SalI-NotI linker (Clontech Co.) wasattached to the ends of the resultant double-stranded cDNA. Then, theends were phospholylated and the product was applied on a sizefractionation column to obtain cDNA with a length more than 500 bp. DNAwas precipitated with ethanol, dissolved in water and ligated to pcDL-SRα296 (Molecular and Cellular Biology, Vol. 8, pp 466-472, 1988) whichhad been cut with a restriction enzyme EcoRI (Takara Shuzo Co.) andtreated with CIAP (calf intestine alkaline phophatase, Takara ShuzoCo.).

[0233] (3) Screening of Expression Library by Means of Binding to OCIF

[0234] An escherichia coli strail, XL2 Blue MRF′ (Toyobo Co., Ltd.), wastransformed using the DNA produced in Example 8 (2) and-cultured onL-Carbenisilin agar (1% trypton, 0.5% yeast extract, 1% NaCl, 60 μg/mlcarbenisilin, 1.5% agar) prepared in a 24-well plastic plates, toproduce about 100 colonies per well. Transformants in each well weresuspended in 3 ml of Terrific Broth ampicillin culture medium (1.2%trypton, 2.4% yeast extract, 0.4% glycerol, 0.017 M KH₂PO₄, 0.072 MK₂HPO₄, 100 μg/ml ampicillin) and cultured at 37° C. overnight withshaking. Cells were collected by centrifugation to prepare plasmid DNAusing a QIAwell kit (QIAGEN Co.). DNA concentration was determined bymeasuring absorbance at 260 nm. DNA was concentrated by precipitatingwith ethanol and dissolved in distilled water to a concentration of 200ng/μl. Five hundred DNA pools, each of which was obtained from about 100colonies were prepared and were used for transfection into COS-7 cells(RIKEN CELL BANK, RCB0539). COS-7 cells were seeded into DMEM containing10% fetal bovine serum in each well of 24-well plates at a cell densityof 8×10⁴ cells/well and cultured overnight at 37° C. in a CO₂ incubator.Next day, the culture medium was removed and the cells were washed withserum-free DMEM culture medium. The above-described plasmid DNA whichwas previously diluted with an OPTI-MEM culture medium (Gibco BRL Co.)and mixed with Lipofectamine (a transfection reagent, manufactured byGibco BRL Co.) according to the protocol supplied with Lipofectamine.After 15 minutes, the mixture was added to the cells in each well. Theamount of Lipofectamine and DNA used were, respectively, 1 μg and 4 μlper well. After 5 hours, the culture medium was removed and 1 ml of DMEMculture medium (Gibco BRL Co.) containing 10% fetal bovine serum wasadded to each well. The plates were incubated for 2-3 days at 37° C. ina CO₂ incubator(5% CO₂). The COS-7 cells transfected and cultured for2-3 days in this manner were washed with a serum-free DMEM culturemedium. Then, 200 μl of a culture medium for the binding assay(serum-free DMEM culture medium containing 0.2% calf serum albumin, 20mM Hepes buffer, 0.1 mg/ml heparin, and 0.02% NaN₃) with 20 ng/ml of¹²⁵I-labeled OCIF added thereto was added to each well. After culturingfor one hour at 37° C. in a CO₂ incubator (5% CO₂) , the cells werewashed twice with 500 μl of a phosphate buffered saline containing 0.1mg/ml heparin. After washing, 500 μl of 0.1 N NaOH solution was added toeach well. The plates were allowed to stand for 10 minutes at roomtemperature to lyse the cells. The amount of ¹²⁵I in each well wasmeasured using a gamma counter (Packard Co.). One DNA pool containingcDNA encoding the protein which specifically binds to OCIF was found byscreening a total of 500 pools. The DNA pool containing the cDNA wasfurther divided, and the above-described transfection and screeningoperations were repeated to isolate the cDNA which encodes the proteinwhich binds to OCIF. The plasmid containing this cDNA was named pOBM291.The Escherichia coli containing this plasmid was deposited with TheNational Institute of Bioscience and Human Technology, Agency ofIndustrial Science and Technology, Biotechnology Laboratory, as pOBM291on May 23, 1997 under the deposition No. FERM BP-5953.

[0235] The methods of labeling OCIF with ¹²⁵I and quantitative analysisof the ¹²⁵I-labeled OCIF by ELISA are shown below. Labeling of OCIF with¹²⁵I was carried out according to the Iodogen method. Twenty μl of 25mg/ml Iodogen-chloroform solution was added to a 1.5 ml Eppendorf tubeand chloroform was evaporated by heating at 40° C., to prepare anIodogen-coated tube. The tube was washed three times with 400 μl of 0.5M sodium phosphate buffer (Na-Pi, pH 7.0), and 5 μl of 0.5 M Na-Pi (pH7.0) was added. Immediately after the addition of 1.3 μl (18.5 MBq) ofNa-¹²⁵I solution (NEZ-033H20, Amersham Co.), 10 μl of 1 mg/ml rOCIFsolution (monomer type or dimer type) was added to the tube. Aftermixing the contents with a voltex mixer, the tube was allowed to standat room temperature for 30 seconds. The solution in the tube wastransferred to a tube to which 80 μl of 10 mg/ml potassium iodide, 0.5 MNa-Pi (pH 7.0) and 5 μl of a phosphate buffered saline containing 5%bovine serum albumin (BSA-PBS) were previously added. After stirring,the mixture was applied to a spin column (1 ml, G-25 fine, manufacturedby Pharmacia Co.) equilibrated with BSA-PBS, and the column wascentrifuging for 5 minutes at 2000 rpm. Four hundred μl of BSA-PBS wasadded to the fraction eluted from the column. After stirring, 2 μl of analiquot of this solution was sampled to measure the radioactivity by agamma counter. The radiochemical purity of the ¹²⁵I-labeled OCIFsolution thus prepared was determined by measuring radioactivityprecipitated by 10% TCA. The biological activity of the ¹²⁵I-labeledOCIF was measured according to the method of WO 96/26217. Theconcentration of the ¹²⁵I-labeled OCIF was determined by the ELISA asfollows. Specifically, 100 μl of 50 mM NaHCO₃ (pH 9.6) in which theanti-OCIF rabbit polyclonal antibody described in WO 96/26217 wasdissolved to a concentration of 2 μg/ml was added to each well of a96-well immuno-plate (MaxiSorp™, a product of Nunc Co.) The plate wasallowed to stand over night at 4° C. After removing the solution bysuction, 300 μl of Block Aces (Snow Brand Milk Products Co.,Ltd.)/phosphate buffered saline (25/75) (B-PBS) was added to each well.The plate was then allowed to stand for two hours at room temperature.After removing the solution by suction, the wells were washed threetimes with phosphate buffered saline containing 0.01% Polysorbate80(P-PBS). Next, 100 μl of B-PBS containing ¹²⁵I-labeled OCIF orstandard OCIF was added to each well. The plate was then allowed tostand for two hours at room temperature. After removing the solution bysuction, each well was washed six times with 200 μl of P-PBS. Onehundred μl of peroxidase-labeled rabbit anti-OCIF polyclonal antibodydiluted with B-PBS was added to each well. The plate was allowed tostand for two hours at room temperature. After removing the solution bysuction, the wells were washed six times with 200 μl of P-PBS. Then, 100μl of TMB solution (TMB Soluble Reagent, High Sensitivity, Scytek Co.)was added to each well. After incubating the plate at room temperaturefor 2-3 minutes, 100 μl of stopping solution (Stopping Reagent, ScytekCo.) was added to each well. Absorbance at 450 nm of each well wasmeasured using a microplate reader. The concentration of ¹²⁵I-labeledOCIF was determined based on a calibration curve drawn using thestandard preparation of OCIF.

[0236] (4) Determination of the Nucleotide Sequence of the cDNA Encodingthe Entire Amino Acid Sequence of OBM

[0237] The nucleotide sequence of the OBM cDNA obtained in the Example8(3) was determined using a Taq DyeDeoxy Terminator Cycle Sequencing kit(a product of Perkin Elmer Co.). Specifically, the nucleotide sequenceof the insert fragment was directly determined using pOBM291 as atemplate. Fragments with a length of about 1.0 kb and 0.7 kb which wereobtained by digesting pOBM291 with a restriction enzyme EcoRI wereinserted into the EcoRI site of plasmid pUC19 (Takara Shuzo Co.) Thenucleotide sequences of these fragments were also determined. Thefollowing primers were used: primer SRR2 which was used to determinenucleotide sequences of DNA fragments inserted into pcDL-SR α296,M13PrimerM3 and M13PrmerRV (both manufactured by Takara Shuzo Co.) whichwere used to determine the nucleotide sequences of DNA fragmentsinserted into plasmid pUC19, and synthesized primer OBM#8 designed basedon the nucleotide sequence of OBMcDNA. Sequences of these primers areshown as the Sequence ID No. 3 to No. 6 in the sequence table.

[0238] In addition, the nucleotide sequence of OBMcDNA determined isshown as Sequence ID No. 2 and the amino acid sequence determinedtherefrom is shown as the Sequence ID No. 1.

Example 9

[0239] Expression of the Protein Encoded by the cDNA of the PresentInvention

[0240] Plasmid pOBM291 was transfected into COS-7 cells in each well ofa 6-well plate using Lipofectamine and the transfected COS-7 cells werecultured for two days in DMEM containing 10% fetal bovine serum. Themedium was replaced with a cysteine-methionine-free DMEM (DainipponSeiyaku Co. Ltd.) (800 μl/well) containing 5% dialyzed fetal bovineserum. The cells were cultured for 15 minutes, followed by the additionof 14 μl of Express Protein Labeling Mix (10 mCi/ml, manufactured by NENCo.). After culturing for four hours, 200 μl of DMEM including 10% fetalbovine serum was added. After one hour culturing, the cells were washedtwice with PBS. Then, 0.5 ml of a TSA buffer (10 mM Tris-HCl (pH 8.0)containing 0.14 M NaCl, 0.025% NaN₃), containing 1% TritonX-100, 1%bovine hemoglobin, 10 μg/ml leupeptin, 0.2 TIU/ml aprotinin, 1 mM PMSF,was added and the mixture was allowed to stand for one hour on ice.Cells were disrupted by pipetting and centrifuged at 3000×g for 10minutes at 4° C. to obtain supernatant. 200 μl of dilution buffer (TSAbuffer containing 0.1% TritonX-100, 0.1% bovine hemoglobin, 10 μg/mlleupeptin, 0.2 TIU/ml aprotinin, 1 mM PMSF) was added to 100 μl of thissupernatant. The mixture was shaken for one hour at 4° C. with protein ASepharose (50 μl). The resultant mixture was centrifuged at 1500×g forone minute at 4° C. to collect supernatant, and thereby fraction(s)which is non-specifically adsorbed to Protein A Sepharose was removed.OCIF (1 μg) was added to this supernatant and the mixture was shaken at4° C. for one hour to achieve the binding of OCIF to OBM. Anti-OCIFpolyclonal antibody (50 μg) was added and the mixture was shaken for onehour at 4° C. Then, Protein A Sepharose (10 μl) was added and themixture was shaken for an additional hour at 4° C., followed bycentrifuge at 1500×g for 1 minute at 4° C. to collect precipitate. Theprecipitate was washed twice with dilution buffer, twice with a bovinehemoglobin-free dilution buffer, once with TSA buffer, and once with 50mM Tris-HCl (pH 6.5). After washing, SDS buffer (0.125 M Tris-HCl, 4%sodium dodecylsulfate, 20% glycerol, 0.002% Bromophenol Blue, pH 6.8)containing 10% β-mercaptoethanol was added to the precipitate. Themixture was heated for 5 minutes at 100° C. and subjected to SDS-PAGE(12.5% polyacrylamide gel, Daiichi Chemical Co., Ltd.). The gel wasfixed according to a conventional method. Isotope signals were amplifiedusing Amplify™ (Amersham Co.) and the sample was exposed to Bio Max MRfilm (KODAK Co.) at −80° C. The results are shown in FIG. 8, whichindicates that the protein encoded by the cDNA of the present inventionhas a molecular weight of about 40,000.

Example 10

[0241] Binding of the Protein Encoded by the cDNA of the PresentInvention to OCIF

[0242] Plasmid pOBM291 was transfected into COS cells in each well of a24-well plate using Lipofectamine. After culturing for 2-3 days, thecells were washed with serum-free DMEM culture medium. 200 μl of culturemedium for the binding assay (serum-free DMEM culture medium containing0.2% calf serum, albumin, 20 mM Hepes, 0.1 mg/ml heparin, and 0.2%NaN₃), supplemented with 20 ng/ml ¹²⁵I-labeled OCIF, was added to thewells. To the other wells, 200 μl of culture medium for the bindingassay to which 8 μg/ml of unlabelled OCIF had been added, in addition to20 ng/ml ¹²⁵-labeled OCIF, was added. After culturing for one hour at37° C. in a CO₂ incubator (5% CO₂), the cells were washed twice with 500μl of phosphate buffered saline containing 0.1 mg/ml of heparin. Then,500 μl of 0.1 N NaOH solution was added to each well and the plate wasallowed to stand for 10 minutes at room temperature to dissolve thecells. The amount of ²⁵¹I in each well was measured by a gamma counter.As a result, as shown in FIG. 9, the ¹²⁵I-labeled OCIF was found to bindonly to the cells in which plasmid pOBM291 was transfected. In addition,the binding was confirmed to be conspicuously inhibited by the additionof (unlabeled) OCIF at a 400-fold concentration. These results havedemonstrated that the protein OBM encoded by the cDNA on plasmid pOBM291specifically binds to OCIF on the surface of the transfected COS-7cells.

Example 11

[0243] Crosslinking of ¹²⁵I-Labeled OCIF and the Protein Encoded by thecDNA of the Present Invention

[0244] Crosslinking of ¹²⁵I-labeled monomer type OCIF and the proteinencoded by the cDNA of the present invention was carried out toinvestigate the characteristics of the protein encoded by the cDNA ofthe present invention in further detail. After transfection of plasmidpOBM291 into COS-7 cells according to the method used in the Example8(3), 200 μl of the culture medium for the binding assay, as describedabove, containing ¹²⁵I-labeled OCIF (25 ng/ml) was added to the wells.The culture medium for the binding assay to which unlabeled OCIF at a400-fold concentration was added in addition to ¹²⁵I-labeled OCIF wasadded to the other wells. After culturing for one hour at 37° C. in aCO₂ incubator(5% CO₂), the cells were washed twice with 500 μl ofphosphate buffered saline containing 0.1 mg/ml heparin. Five hundred μlof phosphate buffered saline containing 100 μg/ml of a crosslinkingagent, DSS (disuccinimidyl suberate, manufactured by Pierce Co.) wasadded to the cells, followed by a reaction for 10 minutes at 0° C. Thecells in these wells were washed twice with 1 ml of cold phosphatebuffered saline (0° C.). After the addition of 100 μl of 20 mM Hepesbuffer containing 1% Triton X-100 (Wako Pure Chemicals Co., Ltd.), 2 mMPMSF (Phenylmethylsulfonyl fluoride, Sigma Co.), 10 μM Pepstatin (WakoPure Chemicals Co., Ltd.), 10 μM Leupeptin (Wako Pure Chemicals Co.,Ltd.), 10 μM antipain (Wako Pure Chemicals Co., Ltd.) and 2 mM EDTA(Wako Pure Chemicals Co., Ltd.), the wells were allowed to stand for 30minutes at room temperature to dissolve the cells. Fifteen μl aliquotsof these samples were heated in the presence of SDS under reducingconditions according to a conventional method and subjected toSDS-electrophoresis using 4-20% polyacrylamide gradient gel (DaiichiPure Chemical Co., Ltd.). After the electrophoresis, the gel was driedand exposed for 24 hours at −80° C. to a BioMax MS film (Kodak Co.)using a BioMax MS sensitization screen (Kodak Co.). The exposed film wasdeveloped according to a conventional method. As a result, a band with amolecular weight of a range of 90,000-110,000, shown in FIG. 10, wasdetected by crosslinking the ¹²⁵I-labeled monomer type OCIF and theprotein encoded by the cDNA of the present invention.

Example 12

[0245] Northern Blotting Analysis

[0246] ST2 cells cultured to become confluent in a 25 cm² T flask forattached-cell cultures were treated with trypsin and stripped from the Tflask. After washing, the cells were seeded into a 225 cm² T flask andcultured for 4 days in a CO₂ incubator with 60 ml of an α-MEM culturemedium containing 10⁻⁸ M active-form vitamin D₃, 10⁻⁷ M dexamethasone,and 10% fetal bovine serum. Total RNA was extracted from the culturedST2 cells using ISOGEN (Wako Pure Chemicals Co., Ltd.). The total RNAwas also extracted in the same manner from ST2 cells which were culturedin the absence of the active-form vitamin D₃ and dexamethasone. Afterthe addition of 2.0 μl of 5× gel electrophoresis buffer solution (0.2 Mmorpholino propane sulfonic acid, pH 7.0, 50 mM sodium acetate, 5 mMEDTA) and 3.5 μl of formaldehyde, and 10.0 μl of formamide to 20 μg (4.5μl) of each of the total RNAs, the mixtures were incubated for 15minutes at 55° C. and subjected to electrophoresis. The gel forelectrophoresis was prepared according to the formulation of 1.0%agarose, 2.2 M deionized formaldehyde, 40 mM morpholinopropane sulfonicacid (pH 7.0), 10 mM sodium acetate, and 1 mM EDTA. The electrophoresiswas carried out in a buffer solution of 40 mM morpholino propanesulfonic acid, pH 7.0, 10 mM sodium acetate, and 1 mM EDTA. After theelectrophoresis, RNA was transferred onto nylon membrane. About 1.0 kbDNA fragment was obtained by digesting pOBM291 with a restrictionenzyme, EcoRI. Hybridization was carried out using this DNA fragment,labeled with a Megaprime DNA labeling kit (Amersham Co.) and α-³²p-dCTP(Amersham Co.), as a probe. As a result, as shown in FIG. 11, it wasconfirmed that when ST2 cells were cultured in the presence ofactive-form vitamin D₃ and dexamethasone, gene expression of the proteinencoded by the cDNA of the present invention (OBM) is induced strongly.

Example 13

[0247] Osteoclasts Formation Supporting Capability of the ProteinEncoded by the cDNA of the Present Invention

[0248] pOBM291 was transfected into COS cells according to the samemethod described in the Example 8(3). After three days, trypsinizedcells were washed once with phosphate buffered saline solution bycentrifugation, then fixed with PBS containing 1% paraformaldehyde atroom temperature for 5 minutes, followed by washing with PBS six timesby centrifugation. 700 μl of 1×10⁶/ml mouse spleen cells and 350 μl of4×10⁴/ml ST2 cells which were suspended in α-MEM culture mediumcontaining 10⁻⁸ M active-form vitamin D₃, 10⁻⁷ M dexamethasone, and 10%fetal bovine serum, were added to a 24-well plate. TC insert (Nunc Co.)was set in each well. The above-described fixed COS cells (350 μl )diluted to various concentrations with the above-mentioned culturemedium and OCIF solution (50 μl) were added to the TC insert andcultured for 6 days at 37° C. As a result, it was confirmed that theosteoclasts formation inhibitive activity of OCIF can be inhibited bythe protein encoded by the cDNA of the present invention.

Example 14

[0249] Expression of Secreted-Form OBM

[0250] (1) Construction of a Plasmid for the Expression of Secreted-FormOBM

[0251] A PCR reaction was carried out using OBM HF (Sequence Table,Sequence ID No. 7) and OBM XR (Sequence Table, Sequence ID No. 8) asprimers and pOBM291 as a template. After purification by agarose gelelectrophoresis, the product was digested with restriction enzymesHindIII and EcoRI, and further purified by agarose gel electrophoresis.The purified fragment (0.6 kb), Hind III/EcoRI fragment (5.2 kb) of pSecTagA (Invitrogen Co.), and EcoRI/PmacI fragment (0.32 kb) of OBM cDNAwere ligated using a a ligation kit ver. 2 (Takara Shuzo Co.).Escherichia coli DH5 α was transformed using the reaction product.Plasmids were purified by means of alkali SDS method from the resultingampicillin resistant strains and digested with restriction enzymes toselect a plasmid with fragments of a length of 0.6 Kb and 0.32 kb beinginserted into pSec TagA. Selected plasmid was identified as having asequence encoding the secreted-form OBM (nucleotide sequence: 338-1355in Sequence ID No. 2, amino acid sequence: 72-316 in the Sequence IDNo. 1) by sequencing using a dyeterminator cycle sequencing FS kit(Perkin Elmer Co.). This plasmid was digested with restriction enzymesNhel and Xhol to isolate a fragment (1.0 kb) containing thesecreted-form OBM cDNA by agarose gel electrophoresis. This fragment wasinserted into the NheI/XhoI fragment (10.4 kb) of an expression vector,pCEP4 (Invitrogen Co.), using a ligation kit and Escherichia coli DH5 αwas transformed using the reaction product thereof. Plasmids werepurified by alkali SDS method from the resulting ampicillin resistantstrains and digested with restriction enzymes to select an Escherichiacoli strain having the secreted-form OBM expression plasmid (pCEP sOBM)with the correct structure. The Escherichia coli strain containing thepCEP sOBM was cultured and pCEP sOBM was purified using QIA filterplasmid midi kit (QIAGEN Co.).

[0252] (2) Expression of Secreted-Form OBM

[0253] 293-EBNA cells were suspended in IMDM containing 10% FCS(IMDM-10% FCS)and seeded into a 24-well plate coated with collagen(manufactured by Sumitomo Bakelite Co., Ltd.) in a cell density of2×10⁵/2 ml/well and cultured overnight. The cells were transfected with1 μg of pCEP sOBM or pCEP4 using 4 μl of Lipofectamine (Gibco Co.).After culturing for two days in 0.5 ml of a serum-free IMDM or IMDM-10%FCS, the conditioned medium was collected. Expression of thesecreted-form OBM in the conditioned medium was confirmed as follows.Sodium hydrogen carbonate was added to the conditioned medium to a finalconcentration of 0.1 M and the solution was added to a 96-well plate.The plate was allowed to stand overnight at 4° C., thereby immobilizingOBM in the conditioned medium on the 96-well plate. The plate was filledwith a Block Ace™ (Snow Brand Milk Products Co., Ltd.) solution dilutedfour-fold with PBS (B-PBS) and allowed to stand for two hours at roomtemperature to block residual binding sites of the plate. After theaddition to each well of 100 μl of 3-100 ng/ml of OCIF which was dilutedwith B-PBS, the plate was allowed to stand for two hours at 37° C.,followed by washing with PBS containing 0.05% Tween 20(PBS-T). Then, 100μl of a peroxidase-labeled rabbit anti-OCIF polyclonal antibodydescribed in WO 96/26217 which was diluted with B-PBS was added to eachwell. After allowing to stand for two hours at 37° C., the wells werewashed six times with PBS-T. Then, a TMB solution (TMB Soluble Reagent,High Sensitivity, Scytek Co.) was added in the amount of 100 μl per welland allowed to stand at room temperature for about 10 minutes, whereuponthe reaction was terminated by the addition of 100 μl of a terminationsolution (Stopping Reagent, Scytek Co.) to each well. Absorbance at 450nm of each well was measured by a microplate reader. The results areshown in FIG. 12 which indicates that the absorbance at 450 nm increasedaccording to the concentration of the added OCIF in the plate in whichthe conditioned medium of the cells transfected with pCEP sOBM wasimmobilized. On the other hand, no increase in absorbance was seen inthe plate in which the conditioned medium of the cells transfected withvector pCEP4 was immobilized. FIG. 13 shows the results of an experimentwherein the proportion of the conditioned medium which is used forimmobilization was changed within a range of 5-90% and a specificconcentration of OCIF (50 ng/ml) was added. It can be seen that theabsorbance at 450 nm increased according to the increase in theproportion of the conditioned medium in the plate wherein theconditioned medium of the cells transfected with pCEPsOBM wasimmobilized, whereas no such increase in absorbance was seen in theplate wherein the conditioned medium of the cells transfected withvector pCEP4 was immobilized. From these results, it was confirmed thatsecreted-form OBM is produced into the conditioned medium of the cellstransfected with pCEP sOBM.

Example 15

[0254] Expression of Thioredoxin-OBM Fusion Protein (Trx-OBM)

[0255] (1) Construction of a Thioredoxin-OBM Fusion Protein (Trx-OBM)Expression Vector

[0256] Ten μl of 10× ExTaq buffer (Takara Shuzo Co.), 8 μl of 10 mM dNTP(Takara Shuzo Co.), 77.5 μl of sterilized distilled water, 2 μl of anaqueous solution of pOBM291 (10 ng/μl), 1 μl of primer OBM3 (100pmol/μl, Sequence Table, Sequence ID No. 9), 1 μl of primer OBMSalR2(100 pmol/μl, Sequence Table, Sequence ID No. 10) and 0.5 μl of ExTaq (5u/μl) (Takara Shuzo Co.) were mixed and reacted (PCR reaction) in amicro centrifugel tube. After reacting at 95° C. for 5 minutes, at 50°C. for one second, at 55° C. for one minute, at 74° C. for one second,and at 72° C. for 5 minutes, a cycle reaction consisting of a reactionat 96° C. for one minute, at 50° C. for one second, at 55° C. for oneminute, at 74° C. for one second, and at 72° C. for 3 minutes, wasrepeated 25 times. From the total reaction liquid DNA fragment of about750 bp was purified by 1% agarose gel electrophoresis using QIAEX II gelextraction kit (QIAGEN Co.). The whole amount of purified DNA fragmentwas digested with restriction enzymes SaII and EcoRI (Takara Shuzo Co.)and subjected to an 1.5% agarose gel electrophoresis to purify a DNAfragment of about 160 bp (Fragment 1), which was dissolved in 20 μl ofsterilized distilled water. In the same manner, a DNA fragment of about580 bp (Fragment 2) obtained by digesting 4 μg of pOBM291 withrestriction enzymes BamHl and EcoRI (Takara Shuzo Co.) and a DNAfragment of about 3.6 kb (Fragment 3) obtained by digesting 2 μg ofpTrXFus (Invitrogen Co.) with restriction enzymes BamHI and SaII (TakaraShuzo Co.) were respectively purified and dissolved in 20 μl ofsterilized distilled water. The QIAEXII gel extraction kit was used forthe purification of DNA fragments. Fragments 1-3 were ligated byincubating at 16° C. for 2.5 hours using DNA ligation kit ver.2 (TakaraShuzo Co.). Using the ligation reaction liquid, Escherichia coli strainGI724 (Invirogen Co.) was transformed according to the method describedin the Instruction Manual of ThioFusion Expression System (InvirogenCo.). A microorganism strain with plasmid in which the OBM cDNA fragment(nucleotide sequence: 350-1111 in the Sequence ID No. 2, amino acidsequence: 76-316 in the Sequence ID No. 1) is fused in frame to athioredoxin gene was selected from the resulting ampicillin resistanttransformants by the analysis of restriction maps obtained by digestionwith restriction enzymes and DNA sequence determination. Themicroorganism strain thus obtained was named GI724/pTrxOBM25.

[0257] (2) Expression of OBM in Escherichia coli

[0258] GI724/pTrxOBM25 and GI724 containing pTrxFus (GI724/pTrxFus) wererespectively cultured six hours with shaking at 30° C. in 2 ml ofRMG-Amp culture medium (0.6% Na₂HPO₄, 0.3% KH₂PO₄, 0.05% NaCl, 0.1%NH₄Cl, 1.2% casamino acid (Difco Co.), 1% glycerol, 1 mM MgCl₂, and 100μg/ml ampicillin (Sigma Co.), pH 7.4). The broth 0.5 ml of the broth wasadded to 50 ml of Induction culture medium (0.6% Na₂HPO₄, 0.3% KH₂PO₄,0.05% NaCl, 0.1% NH₄Cl, 0.2% casamino acid, 0.5% glucose, 1 mM MgCl₂,100 μg/ml ampicillin, pH 7.4) and cultured with shaking at 30° C. WhenOD_(600nm). reached about 0.5, L-tryptophan was added to a finalconcentration of 0.1 mg/ml, followed by shaking the culture at 30° C.for an additional 6 hours. The culture broth was centrifuged at 3000×gto collect the cells, which were suspended in 12.5 ml of PBS (10 mMphosphate buffer, 0.15 M NaCl, pH 7.4). The suspension was subjected toan ultrasonic generator (Ultrasonics Co.) to disrupt the cells. Thedisrupted cells were centrifuged at 7000×g for 30 minutes to obtain asupernatant liquid as a soluble protein fraction. Ten μl of this solubleprotein fraction was subjected to SDS polyacrylamide (10%)electrophoresis under reducing conditions. As a result, a band with amolecular weight of 40 kDa which was not detected in the soluble proteinfraction of GI724/pTrxFus was found in the soluble protein fraction ofGI724/pTrxOBM25 (FIG. 14). Accordingly, it was confirmed that a fusionprotein (Trx-OBM) of thioredoxin and OBM was expressed in Escherichiacoli.

[0259] (3) Binding Capability of Trx-OBM to OCIF

[0260] Binding of the expressed Trx-OBM to OCIF was confirmed accordingto the following experiment. Anti-thioredoxin antibody (Invirogen Co.)which was diluted to 5000-fold with 10 mM sodium hydrogen carbonatesolution was added to a 96-well immunoplate (Nunc Co.) in the amount of100 μl per well. After being allowed to stand overnight at 4° C., theliquid in the wells was discarded. Two hundred μl of a solution preparedby diluting Block Ace™ (Snow Brand Milk Products Co., Ltd.) two-foldwith PBS (BA-PBS) was added to each well. After being allowed to standfor one hour at room temperature,the solution was discarded and solubleprotein fractions originating from the above-described GI724/pTrxOBM25or GI724/pTrxFus, each diluted with BA-PBS in various concentrationswere added to each well in the amount of 100 μl. After being allowed tostand for two hours at room temperature, each well was washed threetimes with PBS-T and charged with 100 μl of OCIF (100 ng/ml) which wasdiluted with BA-PBS. After being allowed to stand for two hours at roomtemperature, each well was washed three times with PBS-T and chargedwith 100 μl of peroxidase- labeled rabbit anti- OCIF polyclonal antibody(described in WO 96/26217) which was diluted 2,000-fold with BA-PBS.After being allowed to stand for two hours at room temperature, eachwell was washed six times with PBS-T and charged with 100 μl of TMBsolution (TMB Soluble Reagent, High Sensitivity, Scytek Co.). Afterbeing allowed to stand for about 10 minutes at room temperature, eachwell was charged with 100 μl of termination solution (Stopping Reagent,Scytek Co.). Absorbance of each well at 450 nm was measured by amicroplate reader. The results are shown in FIG. 15. There was nodifference in absorbance between the sample with the soluble proteinfraction originating from GI724/pTrxFus added thereto and the samplewithout the addition of this soluble protein fraction. On the otherhand, the absorbance increased in the samples to which the solubleprotein fraction originating from GI724/pTrxOBM25 was added inproportion to the concentration of the soluble protein fraction. Theresults of the other experiment wherein the dilution rate of the solubleprotein fraction was maintained constant (1%) while adding OCIF dilutedwith BA-PBS in different concentrations (0-100 ng/ml) are shown in FIG.16. It can be seen that the absorbance remained low at anyconcentrations of OCIF in samples using a soluble protein fractionoriginating from GI724/pTrxFus, whereas the absorbance increased inproportion to the OCIF concentration in the samples to which the solubleprotein fraction originating from GI724/pTrxOBM25 was added. Based onthese results, it was confirmed that Trx-OBM which is produced fromGI724/pTrxOBM25 has a capability of binding to OCIF.

[0261] (4) Large-Scale Cultivation of Escherichia coli which ProducesTrx-OBM

[0262] GI724/pTrxOBM25 cells were spread on RMG-Amp agar (0.6% Na₂PO₄,0.3% KH₂PO₄, 0.05% NaCl, 0.1% NH₄Cl, 2% casamino acid, 1% glycerol, 1 mMMgCl₂, 100 μg/ml ampicillin, 1.5% agar, pH 7.4) using a platinumtransfer loop. The cells were cultured overnight at 30° C. The culturedcells were suspended in 10 ml of Induction medium. The suspension wasadded 5 ml for each to two 2 l Erlenmeyer flasks containing 500 ml ofInduction medium and cultured at 30 ° C. with shaking. When theOD_(600nm) reached about 0.5, L-tryptophan was added to a finalconcentration of 0.1 mg/ml. Culturing with shaking was continued for sixhours at 30° C. The culture broth was centrifuged for 20 minutes at3000×g to collect the cells, which were suspended in 160 ml of PBS. Thesuspension was subjected to an ultrasonic generator (Ultrasonics Co.) todisrupt the cells. The supernatant liquid was centrifuged for 30 minutesat 7000×g to obtain a soluble protein fraction.

[0263] (5) Preparation of OCIF-Immobilized Affinity Column

[0264] Two g of TSKgel AF-Tolesyl Toyopal 650 (Tosoh Corp.) and 40 ml of1.0 M potassium phosphate buffer (pH 7.5) containing 35.0 mg ofrecombinant OCIF, which was prepared according to the method describedin WO 96/26217, were mixed. The mixture was gently shaken overnight at4° C. to effect a coupling reaction. The reaction mixture wascentrifuged to remove the supernatant. To inactivate excess activeresidues, 40 ml of 0.1 M Tris-HCl buffer (pH 7.5) was added to theprecipitated carrier and the mixture was gently shake at roomtemperature for one hour. The carrier in a column was washed with 0.1 Mglycine-HCl buffer (pH 3.3) containing 0.01% Polysorbate 80 and 0.2 MNaCl and 0.1 M sodium citrate buffer (pH 2.0) containing 0.01%Polysorbate 80 and 0.2 M NaCl. The carrier in the column wasequilibrated by charging twice with 10 mM sodium phosphate buffer (pH7.4) containing 0.01% Polysorbate 80.

[0265] (6) Purification of Trx-OBM Using OCIF-Immobilized AffinityColumn

[0266] Unless otherwise indicated, purification of Trx-OBM was carriedout at 4° C. The above-mentioned OCIF-immobilized affinity carrier (10ml) and the soluble protein fraction (120 ml) prepared in Example 15 (4)were mixed. The mixture was gently shaken overnight at 4° C. in four50-ml centrifuge tubes using a rotor. An Econo-column™ (internaldiameter: 1.5 cm, length: 15 cm, manufactured by BioRad Co.) was filledwith the carrier in the mixture. The column was charged with 300 ml ofPBS containing 0.01% Polysorbate 80, 100 ml of 10 mM sodium phosphatebuffer (pH 7.0) containing 0.01% Polysorbate 80 and 2 M NaCl, and 100 mlof 0.1 M glycine-HCl buffer (pH 3.3) containing 0.01% Polysorbate 80 and0.2 M NaCl, in that order. Next, proteins adsorbed in the column wereeluted with 0.1 M sodium citrate buffer (pH 2.0) containing 0.01%Polysorbate 80 and 0.2 M NaCl. The eluate was collected in 5 mlportions. Each fraction thus collected was immediately neutralized withaddition 10% volume of 2 M Tris buffer (pH 8.0). Presence or absence ofTrx-OBM in the eluted fractions was determined according to the methodpreviously described in Example 15(3) (the binding capability to OCIF).The fractions containing Trx-OBM were collected and purified further.

[0267] (7) Purification of Trx-OBM by Gel Filtration

[0268] About 25 ml of Trx-OBM fractions obtained in Example 15 (6) wasconcentrated to about 0.5 ml by centrifuge using Centriplus 10 andCentricon 10 (Amicon Co.). This sample was applied to a Superose 12 HR10/30 column (1.0×30 cm, Pharmacia Co.) previously equilibrated with PBScontaining 0.01% Polysorbate 80. For the separation, PBS containing0.01% Polysorbate 80 was used as a mobile phase at a flow rate of 0.25ml/min. The eluate from the column was collected in 0.25 ml portions.The Trx-OBM in the thus-collected fractions was detected by the samemethod as previously described in Example 15(3) and bySDS-polyacrylamide electrophoresis (10-15% polyacrylamide gel, PharmaciaCo.) using Phast System (Pharmacia Co.) and silver staining. Fractions(Fr. 20-23) containing purified Trx-OBM were collected and the proteinconcentration of Trx-OBM was determined. The measurement of the proteinconcentration was carried out using bovine serum albumin as a standardsubstance using DC-Protein assay kit (BioRad Co.).

Example 16

[0269] Osteoclast Formation-Inducing Activity of OBM

[0270] pOBM291 and pcDL-SR α296 were respectively transfected into COS-7cells using Lipofectamine (Gibco Co.). The cells were cultured in DMEMcontaining 10% FCS for one day, trypsinized, plated on cover slips(15 mmround shape, manufactured by Matsunami Co.) in 24-well plates at 5×10⁴cells per well, and cultured for 2 days. The culture plate was washedonce with PBS. The cells were fixed with PBS containing 1%paraformaldehyde at room temperature for 8 minutes. The plate on whichthe fixed cells were attached was washed 6 times with PBS,then 700 μl ofmouse spleen cells suspended at 1×10⁶/ml in α-MEM containing 10⁻⁸ Mactive-form vitamin D₃, 10⁻⁷ M dexamethasone, and 10% fetal bovine serumwere added to each well. Millicell PCF (Millipore Co.) was set in eachwell and a suspension of ST2 cells in the above-mentioned culture medium(4×10⁴/ml) were added, 700 μl per well, into the Millicell PCF followedby incubation at 37° C. for 6 days. After the culture, the Millicell PCFwas removed, the plate was washed once with PBS, and the cells werefixed with acetone-ethanol solution (50:50) for one minute. Then, cellsexhibiting tartaric acid-resistant acid phophatase activity (TRAP),which is a specific marker for osteoclast, were selectively stainedusing LEUKOCYTE ACID PHOSPHATASE kit (Sigma Co.). As a result ofmicroscopic observation, TRAP-positive cells were not detected in thewells in which COS-7 cells transfected with pcDL-SR α296 were fixed. Incontrast, 45±18 (average±standard deviation, n=3) TRAP positive cellswere observed in the wells in which COS-7 cells transfected with pOBM291were fixed. Moreover, it was also confirmed that calcitonin bound tothese TRAP positive cells. Based on these findings, it has been proventhat OBM has osteoclast formation-inducing activity.

Example 17

[0271] Osteoclast Formation-Inducing Activity of Trx-OBM andSecreted-Form OBM

[0272] Mouse spleen cells were suspended in α-MEM containing 10⁻⁸ Mactive-form vitamin D₃, 10⁻⁷ M dexamethasone, and 10% fetal bovine serumat a concentration of 2×10⁶/ml. The suspension was added to a 24 wellplate in the amount of 350 μl per well. Each well was then charged with350 μl of a solution prepared by diluting purified Trx-OBM with theabove-mentioned culture medium (40 ng/ml), 350 μl of solution preparedby 10-fold diluting conditioned medium which was produced by culturing293-EBNA cells, in which pCEP sOBM or pCEP4 were transfected, inIMDM-10% FCS, with the above-mentioned culture medium, or 350 μl only ofthe above-mentioned culture medium. The Millicell PCF (Mollipore Co.)was set on each well, to which 600 μl of ST2 cells which was suspendedin the above-mentioned culture medium (4×10⁴/ml) were added. Aftercultured for six days, Millicell PCF was removed. The plate was washedonce with PBS and the cells were fixed with acetone-ethanol solution(50:50) for one minute. Then, the cells exhibiting the tartaric acidresistant acidic phophatase activity (TRAP activity) were selectivelystained using LEUKOCYTE ACID PHOSPHATASE kit (Sigma Co.). The result ofmicroscopic observation revealed that no cells exhibiting the TRAPactivity were detected in the wells to which no Trx-OBM was added,whereas 106±21 (average±standard deviation, n=3) TRAP-positive cellswere observed in the wells to which Trx-OBM was added. Similarly, whileno cells exhibiting TRAP activity were detected in the wells to whichconditioned medium of 293-EBNA transfected with pCEP4 had been added,120±31 (average±standard deviation, n=3) TRAP positive cells wereobserved in the wells to which conditioned medium of 293-EBNAtransfected with pCEPsOBM had been added. Moreover, it was alsoconfirmed that calcitonin binds to these TRAP positive cells. Theseresults have proven that Trx-OBM and secreted-form OBM exhibitosteoclast formation-inducing activity.

Example 18

[0273] Identity of the Protein OBM Expressed by the cDNA of the PresentInvention and the Natural Type OCIF-Binding Protein of the PresentInvention

[0274] (1) Preparation of Rabbit Anti-OBM Polyclonal Antibody

[0275] Three male Japanese white rabbits (weight: 2.5-3.0 kg, suppliedby Kitayama Labes Co.) were immunized with the purified OBM(thioredoxin-OBM fusion protein) produced according to the method inExamples 14(6) and 14(7) by subcutaneously injecting 1 ml/dose ofemulsion prepared by mixing 200 μg/ml of the purified OBM with equalvolume of Freund's complete adjuvant (DIFCO Co.), six times, once aweek. Ten days after the last immunization, the rabbits wereexsanguinated. Antibody was purified from the serum as follows. Ammoniumsulfate was added to the antiserum which was diluted two-fold with PBSto a final concentration of 40% (w/v %) . After being allowed to standfor one hour at 4° C., the mixture was centrifuged for 20 minutes at8000×g to obtain a precipitate. The precipitate was dissolved in a smallamount of PBS, dialyzed against PBS at 4° C., and loaded to a ProteinG-Sepharose column (manufactured by Pharmacia Co.) After washed withPBS, the adsorbed immunoglobulin G was eluted with 0.1 M glycine-HClbuffer solution (pH 3.0). The eluate was immediately neutralized with1.5 M Tris-HCl buffer (pH 8.7). After dialyzing the eluted proteinfractions against PBS, the absorbance at 280 nm was measured todetermine the protein concentration (E^(1%) 13.5). Anti-OBM antibodylabeled with horseradish peroxidase was prepared using amaleimide-activated paroxidase kit (Pierce Co.) as follows. 80 μg ofN-succinimide-S-acetyl thioacetic acid was added to 1 mg of the purifiedantibody and reacted at room temperature for 30 minutes. Five mg ofhydroxylamine was added to the resulting mixture to deacetylate theantibody. The modified antibody was fractionated using a polyacrylamidedesalting column. The protein fractions were mixed with 1 mg ofmaleimide-activated peroxidase and reacted for one hour at roomtemperature to obtain enzyme-labeled antibody.

[0276] (2) Capability of Rabbit Anti-OBM Polyclonal Antibody to InhibitSpecific Binding of the Protein (OBM) Expressed by the cDNA of thePresent Invention or the Natural Type Protein of the Present Inventionwith OCIF

[0277] Purified OBM (thioredoxin-OBM fused protein) obtained accordingto the method described in the Examples 15(6) and 15(7) and the naturaltype purified OCIF-binding protein of the Example 2(4) were dissolvedrespectively in 0.1 M sodium carbonate buffer to a concentration of 2μg/ml. An aliquot of each solution was added 100 μl per wellrespectively to a 96-well immunoplate (manufactured by Nunc Co.). Theplate was allowed to stand overnight at 4° C. 200 μl of 50% Block Acewas added to each well and the plate was allowed to stand at roomtemperature for one hour. After washing each well three times with PBScontaining 0.1% Polysolbate 20 (P20-PBS), 100 μl of rabbit anti-OBMantibody solution which was dissolved in 25% Block Ace prepared withP20-PBS to a concentration of 200 μg/ml or 100 μl of 25% Block Ace(containing no antibody) was added to each well, followed by incubationat 37° C. for one hour. Each well was washed three times with P20-PBSand charged with 100 μl/well of a binding test solution (P20-PBScontaining 0.2% calf serum albumin, 20 mM Hepes, and 0.1 mg/ml heparin)to which 20 ng/ml of ¹²⁵-labeled OCIF described in the Example 8(3) wasadded. Alternatively, each well was charged with 100 μl/well of anotherbinding test solution containing 8 μg/ml of unlabeled OCIF in additionto 20 ng/ml ¹²⁵I-labeled OCIF. After incubating these immunoplates at37° C. for one hour, the wells were washed with P20-PBS six times. Theamount of ¹²⁵I in each well was measured by a gamma counter. The resultsare shown in FIG. 17. As shown in the figure, both the purified OBMexpressed using the cDNA of the present invention and the protein thatspecifically bind the natural type OCIF-specifically binding protein ofthe present invention do not bind to the ¹²⁵I-labeled OCIF at all, whenthey were treated with the rabbit anti-OBM polyclonal antibody, whereasboth proteins bound ¹²⁵I-labeled OCIF when untreated with the antibody.The binding of both proteins to ¹²⁵I-labeled OCIF was confirmed to beclearly specific, because those bindings are significantly inhibited bythe addition of 400-fold concentration of unlabelled OCIF (8 μg/ml).Based on the results described above, the rabbit anti-OBM polyclonalantibody recognizes both the OBM which is the protein expressed usingthe cDNA of the present invention and the natural-type OCIF-bindingprotein of the present invention, and it inhibits the specific bindingof these proteins with OCIF.

Example 19

[0278] Cloning of Human OBM cDNA

[0279] (1) Preparation of Mouse OBM Primer

[0280] The mouse OBM primers prepared according to the method of theExamples (OBM#3 and OBM#8) described above, were used for screening ofhuman OBM cDNA. The sequences are shown in the Sequence Table, SequenceID No. 9 and No. 6, respectively.

[0281] (2) Isolation of Human OBM cDNA Fragment by PCR

[0282] Human OBM cDNA fragments were obtained by PCR using the mouse OBMcDNA primers prepared in (1) above and Human Lymph Node Marathon readycDNA (Clontech Co.) as a template. The conditions for PCR were shown asfollows: 10 × EX Taq buffer (Takara Shuzo Co.) 2 μl 2.5 mM dNTP 1.6 μlcDNA solution 1 μl EX Taq (Takara Shuzo Co.) 0.2 μl Distilled water 14.8μl 40 μM primer OBM#3 0.2 μl 40 μM primer OBM#8 0.2 μl

[0283] These solutions were mixed in a microfuge tube and pre-incubatedat 95° C. for 2 minutes, followed by 40 cycles of a three-stage reactionconsisted of reactions at 95° C. for 30 seconds, at 57° C. for 30seconds, and at 72° C. for 2.5 minutes. After the reaction, the solutionwas incubated for 5 minutes at 72° C. and a portion of the solution wassubjected to electrophoresis on an agarose gel. A DNA fragment (about690 bp) amplified by the mouse OBM cDNA primers described above wasdetected.

[0284] (3) Purification of the Human OBM cDNA Fragment Amplified by PCRand Determination of the Nucleotide Sequence

[0285] The human OBM cDNA fragment obtained in the Example 19(2) wasseparated by-electrophoresis on an agarose gel and further purifiedusing a QIAEX gel extraction kit (Qiagen Co.). PCR was again performedusing the purified human OBM cDNA fragment as a template and the mouseOBM cDNA primers described above, to produce a large quantity of thehuman OBM cDNA fragment. The DNA fragment was purified by a QIAEX gelextraction kit in the same manner as above. The nucleotide sequence ofthe purified human OBM cDNA fragment was determined using Taq Dye DeoxyTerminator Cycle Sequencing FS kit (Perkin Elmer Co.) using OBM#3 orOBM#8 (Sequence ID No. 9 and No. 6 respectively) as a primer. Whencompared with the sequence of corresponding area of the mouse OBM cDNA,the nucleotide sequence of the human OBM cDNA fragment showed 80.7%homology with that of the mouse OBM cDNA.

[0286] (4) Screening of a Full-Length Human OBM cDNA by HybridizationUsing the Human OBM cDNA Fragment (About 690 bp) as a Probe

[0287] A full-length human OBM cDNA was screened using the human OBMcDNA fragment (about 690 bp) that was purified in the Example 19(3)andlabeled with [α³²p] dCTP using a Megaprime DNA Labeling kit (AmershamCo.). Human Lymph Node 5′-STRETCH PLUS cDNA library (Clontech Co., theU.S.A) was screened using the DNA probe. According to the manufacturer'sprotocol, Escherichia coli C600 Hfl was infected with the recombinantphage for 15 minutes at 37° C. The infected Escherichia coli was addedto an LB agar (1% trypton, 0.5% yeast extract, 1% NaCl, 0.7% agar) whichwas heated at 45° C. The LB agar was poured onto an LB agar platecontaining 1.5% agar. After overnight incubation at 37° C., HyBond-N™(Amersham Co.) was placed to the plate on which plaques were producedand stored for about 3 minutes. According to a conventional method, thefilter was treated with alkaline solution, neutralized, and dipped in2×SSC solution. DNA was then immobilized onto the filter using the UVCROSSLINKER (Stratagene Co.). The resulting filter was dipped intoRapid-hyb buffer (Amersham Co.). After pretreatment for 15 minutes at65° C., the filter was placed in Rapid-hyb buffer containing theheat-denatured human OBM cDNA fragment (about 690 bp, 5×10⁵ cpm/ml)described above. After overnight hybridization at 65° C., the filter waswashed with 2×SSC, 1×SSC, and 0.1×SSC, each containing 0.1% SDS, in thisorder respectively for 15 minutes at 65° C. Several positive clonesobtained were further purified by repeating the screening twice. A clonepossessing an insert (about 2.2 kb) was selected from the purifiedclones and was used in the following experiments. This purified phagewas named λhOBM. About 10 μg of DNA was obtained from the purified λhOBMusing a QIAGEN Lambda kit (Qiagen Co.) according to the manufacturer'sprotocol. The DNA was digested with restriction enzyme SaII andsubjected to electrophoresis on an agarose gel to separate the hOBMinsert cDNA(about 2.2 kb). This DNA fragment purified using the QIAEXgel extraction kit (Qiagen Co.) was digested with restriction enzymeSaII and inserted into plasmid pUC19 (MBI Co.) which was previouslydigested with a restriction enzyme SaII and dephosphorylated, using aDNA ligation kit ver. 2 (Takara Shuzo Co.). Escherichia coli DH 5α(Gibco BRL Co.) was transformed with the pUC19 containing the resultingDNA fragment. The resulting transformant was named pUC19hOBM. Thetransformant was grown and pUC19hOBM in which the human OBM cDNA (about2.2 kb) was inserted and purified by a conventional method.

[0288] (5) Determination of Nucleotide Sequence of cDNA Encoding theEntire Amino Acid Sequence of Human OBM

[0289] The nucleotide sequence of the resulting human OBM cDNA obtainedin Example 19(4) was determined using the Taq Dye Deoxy Terminator CycleSequencing FS kit (Parkin Elmer Co.). Specifically, the nucleotidesequence of the inserted fragment was determined using pUC19hOBM as atemplate. As primers, primers for the determination of the nucleotidesequence of the inserted fragment DNA in pUC19hOBM, M13 Primer M3 andM13 Primer RV (manufactured by Takara Shuzo Co.), and a syntheticprimer, human OBM#8, designed based on the nucleotide sequence of thehuman OBM cDNA fragment (about 690 bp) were used.

[0290] The nucleotide sequence of the primers used, M13 Primer M3 andM13 Primer RV, are respectively shown as the Sequence ID No. 4 and No.5. The amino acid sequence of human OBM deduced from the nucleotidesequence of human OBM cDNA is shown in the Sequence Table as Sequence IDNo. 11. The nucleotide sequence of human OBM cDNA is shown as SequenceID No. 12.

[0291] The Escherichia coli which was transformed by the pUC19hOBM,which is the plasmid containing the resulting human OBM cDNA, wasdeposited in National Institute of Bioscience and Human Technology,Agency of Industrial Science and Technology, on Aug. 13, 1997 asdeposition No. FERM BP-6058.

Example 20

[0292] Radioiodination of OCIF with ¹²⁵I and Quantitative Analysis of¹²⁵I-Labled OCIF by ELISA

[0293] OCIF was labeled with ¹²⁵I using the IODO-GEN method. Twenty μlof 2.5 mg/ml IODO-GEN-chloroform solution was transferred to a 1.5 mlEppendorf tube and the chloroform was evaporated at 40° C., therebyproviding a tube coated with IODO-GEN. The tube was washed three timeswith 400 μl of 0.5 M sodium phosphate buffer solution (Na-Pi, pH 7.0),followed by the addition of 5 μl of 0.5 M Na-Pi (pH 7.0). To this tubewas added 1.3 μl (18.5 MBq) of Na-¹²⁵I solution (Amersham Co.,NEZ-033H), immediately followed by the addition of 10 μl of 1 mg/ml OCIFsolution (monomer type or dimer type). The mixture was mixed in a voltexmixer and allowed to stand at room temperature for 30 seconds. Thissolution was transferred to a tube to which 80 μl of 0.5 M Na-Pi (pH7.0) solution containing 10 mg/ml potassium iodine and 5 μl of aphosphate buffered saline solution containing 5% bovine serum albumin(BSA-PBS) were previously added. The solution was mixed, applied to aspin column (1 ml, G-25 Sephadex fine, manufactured by Pharmacia Co.)which was equilibrated with BSA-PBS in advance, and centrifuged for 5minutes at 2,000 rpm. Four hundred μl of BSA-PBS was added to thefractions eluted from the column. After mixing, 2 μl of the solution wasused to measure the radioactivity by a gamma counter. The radiochemicalpurity of the ¹²⁵I-labeled OCIF solution obtained above was measured bycounting the radioactivity of fractions precipitated by 10%trichloroacetic acid (TCA).

[0294] The biological activity of the ¹²⁵I-labeled OCIF was measuredaccording to the method described in WO 96/26217. The concentration ofthe ¹²⁵I-labeled OCIF was measured using the ELISA method as follows.Specifically, 50 mM NaHCO₃ (pH 9.6) in which rabbit anti-OCIF polyclonalantibody described in the WO 96/26217 was dissolved to a concentrationof 2 μg/ml was added to each well of a 96-well immunoplate (MaxiSorp™,manufactured by Nunc Co.) in the amount of 100 μl per well. After thesewells were allowed to stand overnight at 4° C., solution was removed.Then the wells were charged with a mixed aqueous solution of Block Ace™(Snow Brand Milk Products Co., Ltd.) and a phosphate buffered salinesolution (25:75) (B-PBS) in the amount of 200 μl/well. The plate wasthen allowed to stand for two hours at room temperature. After thesolution was removed, the wells were washed three times with a phosphatebuffered saline solution containing 0.01% Polysolvate 80 (P-PBS). Next,B-PBS containing ¹²⁵I-labeled OCIF sample or the standard OCIF was addedin the amount of 100 μl/well. The plate was then allowed to stand fortwo hours at room temperature. After the solution was removed, each wellwas washed six times with 200 μl of P-PBS. A solution prepared bydiluting peroxidase-labeled rabbit anti-OCIF polyclonal antibody withB-PBS was added in the amount of 100 μl/well. The plate was allowed tostand for two hours at room temperature. After the solution was removed,the wells were washed six times with 200 μl of P-PBS. Then, a TMBsolution (TMB Soluble Reagent, High Sensitivity, Scytek Co.) was addedin the amount of 100 μl/well. After being allowed to stand at roomtemperature for 2-3 minutes, 100 μl of a termination solution (StoppingReagent, Scytek Co.) was added to each well. Absorbance of each well wasmeasured at 450 nm using a microplate reader. The concentration of¹²⁵-labeled OCIF was determined with a calibration curve prepared usingthe standard OCIF.

Example 21

[0295] Expression of the Protein Encoded by cDNA of the PresentInvention

[0296] (1) Construction of hOBM Expression Vector for Animal Cells

[0297] pUChOBM was digested with restriction enzyme SaII and a DNAfragment (about 2.2 kb) were purified by electrophoresis on an 1%agarose gel. The ends of theDNA fragments were blunted using a DNAblunting kit (Takara Shuzo Co.) (blunted hOBMcDNA fragment). Expressionplasmid pcDL-SR α296 (Molecular and Cellar Biology, Vol. 8, pp 466-472(1988)) was digested with restriction enzyme EcoRI, blunted withblunting kit and ligated with the blunted hOBM cDNA fragment using DNAligation kit ver. 2. Escherichia coli DH α was transformed with theligation reaction. A plasmid in the resulting ampicillin resistanttransformant was subjected to digestion with restriction enzyme toanalyze the DNA restriction map and determine the DNA sequence. As aresult, a strain having a plasmid in which hOBM cDNA is inserted in thesame direction of transcription as that of SR α promotor was selected.The microorganism strain was named DH5 α/phOBM.

[0298] (2) Expression of Human OBM in COS-7 Cells

[0299]Escherichia coli DH5 α/phOBM was cultured and plasmid phOBM waspurified using Qiafilter Plasmid Midi kit (Qiagen Co.) phOBM wastransfected using Lipofectamine into COS-7 cells in the wells of a6-well plate and cultured for two days in DMEM containing 10% fetalbovine serum. The culture medium was replaced withcysteine-methionine-free DMEM (manufactured by Dainippon Seiyaku Co.,Ltd.) to which 5% dialysed fetal bovine serum was added (88 μl/well).The cells were incubated for 15 minutes, followed by addition of 14 μlof Express Protein Labeling Mix (NEN Co., 10 mCi/ml). After four hoursincubation, 200 μl of DMEM containing 10% fetal bovine serum was addedto each well. The cells were cultured for one hour and washed twice withPBS. Then, 0.5 ml of a TSA buffer (10 mM Tris-HCl containing 0.14 M NaCland 0.025% NaN₃, pH 8.0) containing 1% Triton X-100, 1% bovinehemoglobin, 10 μg/ml leupeptin, 0.2 TIU/ml aprotinin, and 1 mM PMSF wasadded to each well and the mixtures were allowed to stand for one houron ice. The cells were mixed by pipetting and centrifuged at 3,000×g,for 10 minutes at 4° C., to obtain supernatants. Two hundred μl of adilution buffer (TSA buffer containing 0.1% Triton X-100, 0.1% bovinehemoglobin, 10 μg/ml leupeptin, 0.2 TIU/ml aprotinin, and 1 mM PMSF) wasadded to 100 μl of the supernatant from each well. The resultingmixtures were agitated at 4° C. for one hour together with Protein ASepharose (50 μl) and centrifuged at 1,500×g for one minute at 4° C., tocollect supernatants, thereby removing the protein whichnon-specifically adsorbed Protein A Sepharose. OCIF (1 μg) was added tothe supernatants and the mixtures were agitated for one hour at 4° C. tobind human OBM and OCIF. Then, rabbit anti-OCIF polyclonal antibody (50μg) was added, followed by agitation at 4° C. for one hour. Protein ASepharose (10 μl) was added to the resulting solution, followed byagitation at 4° C. for an additional hour. The mixtures thus obtainedwere centrifuged for 1 minute at 1,500×g at 4° C. to collectprecipitates. The precipitates were washed twice with a dilution buffer,twice with bovine hemoglobin-free dilution buffer, once with TSA buffer,and once with 50 mM Tris-HCl (pH 6.5). After addition of SDS buffercontaining 10% β-mercaptoethanol (0.125 M Tris-HCl, 4% sodiumdodecylsulfate, 20% glycerol, 0.002% Bromophenol Blue, pH 6.8), themixture was heated for 5 minutes at 100° C. and subjected to SDS-PAGE(12.5% polyacrylamide gel, Daiichi Pure Chemical Co.). The gel was fixedand dried according to a conventional method. After isotope signals wereenhanced using Amplify™ (Amersham Co.), the dried gel was subjected toautoradiography at −80° C. using Bio Max MR film (Kodak Co.). Theresults are shown in FIG. 18, which shows that the molecular weight ofthe protein encoded by the cDNA of the present invention is about40,000.

Example 22

[0300] Binding of the Protein Encoded by cDNA of the Present Inventionand OCIF

[0301] PhOBM, which was purified in the same manner as in the Example21(2), was transfected into COS-7 cells in each well of a 24-well plateusing Lipofectamine. After cultured for 2 to 3 days, the cells werewashed with serum-free DMEM. Two hundred μl of a-culture medium for abinding test medium (serum-free DMEM to which 0.2% bovine serum albumin,20 mM Hepes buffer solution, 0.1 mg/ml heparin, and 0.2% NaN₃were added)containing 20 ng/ml of ¹²⁵I-labeled OCIF was added to the wells. To theother wells, 200 μl of culture medium for the binding test mediumcontaining 8 μg/ml of unlabeled OCIF in addition to 20 ng/ml of¹²⁵I-labeled OCIF, was added. After incubation for one hour at 37° C. ina CO₂ incubator (5% CO₂) , the cells were washed twice with 500 μl of aphosphate buffered saline solution containing 0.1 mg/ml of heparin.Then, 500 μl of 0.1 N NaOH solution was added to each well and the platewas allowed to stand for 10 minutes at room temperature to dissolve thecells. The radioactivity of ¹²⁵I in the wells was measured by a gammacounter. As a result, as shown in FIG. 19, it was confirmed that the¹²⁵I-labeled OCIF binds only to the cells transfected with phOBM.Moreover, the binding was significantly inhibited by adding 400-foldexcess unlabelled OCIF (8 μg/ml). Based on the results described above,the protein, human OBM encoded by the cDNA in the phOBM was confirmed tospecifically bind to OCIF on the surface of COS-7 cells.

Example 23

[0302] Crosslinking of ²⁵¹I-labeled OCIF and the Protein Encoded by thecDNA of the Present Invention

[0303] Crosslinking of ¹²⁵I-labeled monomer type OCIF and the proteinencoded by the cDNA of the present invention was carried out to furtherinvestigate the characteristics of the protein encoded by the cDNA ofthe present invention. After constructing expression vector phOBM andtransfecting into COS-7 cells according to the method used in theExamples 21(1) and 21(2), 200 μl of binding test medium containing¹²⁵I-labeled OCIF (25 ng/ml) described above was added. The binding testmedium to which unlabeled OCIF was added at a 400-fold concentration inaddition to ¹²⁵I-labeled OCIF was used for the other wells. Aftercultured for one hour at 37° C. in a CO₂ incubator(5% CO₂), the cellswere washed twice with 500 μl of phosphate buffered saline containing0.1 mg/ml heparin. Five hundred μl of phosphate buffered saline in which100 μg/ml of a crosslinking agent (DSS: disuccinimidyl suberate,manufactured by Pierce Co.) was dissolved was added to the cells,followed by incubation for 10 minutes at 0° C. The cells in these wellswere washed twice with 1 ml of ice-cold phosphate buffered saline. Afteran addition of 100 μl of 20 mM Hepes buffer solution containing 1%Triton X-100 (Wako Pure Chemicals Co., Ltd.), 2 mM PMSF(Phenylmethylsulfonyl fluoride, Sigma Co.), 10 μM Pepstatin (Wako PureChemicals Co., Ltd.), 10 μM leupeptin (Wako Pure Chemicals Co., Ltd.),10 μM antipain (Wako Pure Chemicals Co., Ltd.) and 2 mM EDTA (Wako PureChemicals Co., Ltd.), the wells were allowed to stand for 30 minutes atroom temperature to dissolve the cells. These samples (15 μl aliquots)were treated with SDS under reducing conditions according to aconventional method and subjected to SDS-electrophoresis using 4-20%polyacrylamide gradient gel (Daiichi Pure Chemical Co., Ltd.). Afterelectrophoresis, the gel was dried and subjected to autoradiography for24 hours at −80° C. using BioMax MS film (Kodak Co.) and BioMax MSsensitization screen (Kodak Co.). The film subjected to autoradiographywas developed according to a conventional method. As a result, a band ofa molecular weight in the range of 90,000-110,000, shown in FIG. 20, wasdetected by crosslinking of ¹²⁵I-labeled monomer type OCIF and theprotein encoded by the cDNA of the present invention.

Example 24

[0304] Expression of Secreted-Form Human OBM

[0305] (1) Construction of Secreted-Form Human OBM Expression Plasmid

[0306] A PCR was carried out using human OBM SF (Sequence Table,Sequence ID No. 13) and mouse OBM #8 (Sequence Table, Sequence ID No. 6)as primers and pUC19hOBM as a template. After purified byelectrophoresis on an agarose gel, the product was digested withrestriction enzymes SPlI and HindIII, and further purified byelectrophoresis on an agarose gel to obtain a purified fragment (0.27kb). Human OBM cDNA was partially digested with restriction enzyme DraIand DNA fragments digested with DraI at one site were purified byelectrophoresis on an agarose gel. The purified fragment was furtherdigested with restriction enzyme HindIII. The 0.53 kb DraI/HindIIIfragment was purified by electrophoresis on an agarose gel. The purifiedfragment was ligated with the 0.27 kb SplI/HindIII fragment derived fromthe PCR described above using ligation kit ver. 2 (Takara Shuzo Co.)together with HindIII/EcoRI fragment (5.2 kb) of pSec TagA (InvirogenCo.). Escherichia coli DH5 α was transformed using the reaction product.Plasmids were purified by alkali SDS method from the resultingampicillin resistant transformants and digested with restriction enzymesto select a plasmid containing 0.27 kb and 0.53 kb-fragments as insertsin pSec TagA. This plasmid was confirmed to have a sequence encoding thesecreted human OBM by sequencing using Tag dyedeoxyterminator cyclesequencing FS kit (Perkin Elmer Co.). The plasmid was digested withrestriction enzymes Nhel and Xhol to prepare a fragment (0.8 kb)corresponding to the secreted human OBM cDNA by electrophoresis on anagarose gel. This fragment was inserted into the NheI and XhoI fragment(10.4 kb) of an expression vector pCEP4 (Invirogen Co.) using ligationkit and Escherichia coli DH5α was transformed using the reactionproduct. Plasmids were purified by alkali-SDS method from the resultingampicillin resistant transformants and digested with restriction enzymesto select an Escherichia coli having the expression plasmid forsecreted-form human OBM (pCEPshOBM). The Escherichia coli containing thepCEPshOBM was cultured and pCEPshOBM was purified using Qiafilterplasmid midi kit (Qiagen Co.).

[0307] (2) Expression of Secreted-Form OBM

[0308] 293-EBNA cells were suspended in IMDM containing 10% FCS(IMDM-10% FCS), added into a 24-well plate coated with collagen(manufactured by Sumitomo Bakelite Co., Ltd.) in a cell density of2×10⁵/2 ml/well and cultured overnight. The cells were transfected with1 μg of pCEPshOBM or pCEP4 using 4 μl of Lipofectamine (Gibco Co.).After cultured for two days in 0.5 ml of a serum-free IMDM or IMDM-10%FCS, the culture supernatants were collected. Expression of the secretedhuman OBM in the culture supernatant was detected as follows. Sodiumbicarbonate was added to the culture supernatants to a finalconcentration of 0.1 M and the mixtures were added to a 96-well plate.The plate was allowed to stand overnight at 4° C., thereby immobilizinghuman OBM in the culture supernatants on the 96-well plate. The platewas blocked using Block Ace™ (Snow Brand Milk Products Co., Ltd.)solution four-fold diluted with PBS (B-PBS) and allowed to stand for twohours at room temperature. After adding 3-100 ng/ml of OCIF which wasdiluted with B-PBS to each well, the plate was allowed to stand for twohours at 37° C., followed by wash with PBS containing 0.05% Polysolvate20 (P-PBS). Then, 100 μl of a peroxidase-labeled rabbit anti-OCIFpolyclonal antibody described in WO 96/26217 which was diluted withB-PBS was added to each well. After allowing to stand for two hours at37° C., the wells were washed six times with P-PBS. Then, TMB solution(TMB Soluble Reagent, High Sensitivity, Scytek Co.) was added in theamount of 100 μl per well and the mixture was allowed to stand at roomtemperature for about 10 minutes. The reaction was terminated by theaddition of 100 μl of termination solution (Stopping Reagent, ScytekCo.) to each well. Absorbance at 450 nm for each well was measured by amicroplate reader. The results are shown in FIG. 21, which indicatesthat the absorbance at 450 nm increased according to the concentrationof the added OCIF in the plate in which the conditioned medium of thecells transfected with pCEPshOBM was immobilized. On the other hand, noincrease in absorbance was seen in the wells in which the conditionedmedium of the cells transfected with vector pCEP4 was immobilized. FIG.22 shows the results of an experiment wherein the proportion of theconditioned medium used for immobilization was changed within a range of5-90% in the presence of a constant concentration of OCIF (50 ng/ml).The absorbance at 450 nm increased according to the increase in theproportion of the conditioned medium in the plate wherein theconditioned medium of the cells transfected with pCEPshOBM wasimmobilized, whereas no such increase in absorbance was seen in theplate wherein the conditioned medium of the cells transfected withvector pCEP4 was immobilized. From these results, it was confirmed thatsecreted-form human OBM is produced in the conditioned medium of thecells transfected with pCBPshOBM.

Example 25

[0309] Expression of Thioredoxin-Human OBM Fusion Protein (Trx-hOBM)

[0310] (1) Construction of a Thioredoxin-Human OBM Fusion Protein(Trx-hOBM) Expression Vector

[0311] Ten μl of 10× ExTaq buffer (Takara Shuzo Co.), 8 μl of 10 mM dNTP(Takara Shuzo Co.), 77.5 μl of sterilized distilled water, 2 μl of anaqueous solution of pUC19hOBM(10 ng/μl), 1 μl of primer, mouse OBM #3(100 pmol/μl, Sequence Table, Sequence ID No. 9), 1 μl of primer, hOBMSalR2 (100 pmol/μl, Sequence Table, Sequence ID No. 14), and 0.5 μl ofExTaq (5 u/μl) (Takara Shuzo Co.) were mixed and reacted (PCR) in amicro centrifugel tube. After the reaction at 95° C. for 5 minutes, at50° C. for one second, at 55° C. for one minute, at 74° C. for onesecond, and at 72° C. for 5 minutes, a cycle reaction consisting of areaction at 96° C. for one minute, at 50° C. for one second, at 55° C.for one minute, at 74° C. for one second, and at 72° C. for 3 minutes,was repeated 25 times. From the total reaction mixture DNA fragment (750bp) was purified. The whole amount of purified DNA fragment was digestedwith restriction enzymes SaII (Takara Shuzo Co.) and BspHI (New EnglandBilabs Co.), and subjected to electrophoresis on a 1% agarose gel toobtain purified DNA fragment (Fragment 1, about 320 bp). The fragmentwas dissolved in 20 μl of sterilized distilled water. In the samemanner, DNA fragment (Fragment 2, about 450 bp) obtained by digesting 4μg of pUC19hOBM with restriction enzymes BamHI, and BspHI (Takara ShuzoCo.) and DNA fragment (Fragment 3, about 3.6 kb), obtained by digesting2 μg of pTrXFus (InVitrogen Co.) with restriction enzymes BamHI, andSaII (Takara Shuzo Co.) were respectively purified and dissolved in 20μl of sterilized distilled water. The QIAEXII gel extraction kit wasused for purification of the DNA fragments. Fragments 1-3 were ligatedby incubating at 16° C. for 2.5 hours using DNA ligation kit ver.2(Takara Shuzo Co.). Using the ligation reaction, Escherichia coli GI724(Invirogen Co.) was transformed according to the method described in theInstruction Manual of ThioFusion Expression System (Invirogen Co.). Amicroorganism strain with plasmid in which the hOBM cDNA fragment isfused in frame to thioredoxin gene was selected from the resultingampicillin resistant transformants by analysis of DNA restriction mapobtained by digestion with restriction enzyme and by determination ofDNA sequence. The microorganism strain thus obtained was namedGI724/pTrxhOBM.

[0312] (2) Expression of Trx-hOBM in Escherichia coli

[0313] GI724/pTrxhOBM and GI724 containing pTrxFus (GI724/pTrxFus) wererespectively cultured six hours with shaking at 30° C. in 2 ml ofRMG-Amp medium (0.6% Na₂HPO₄, 0.3% KH₂PO₄, 0.05% NaCl, 0.1% NH₄Cl, 2%casamino acid, 1% glycerol, 1 mM MgCl₂, 100 μg/ml ampicillin, pH 7.4).The broth(0.5 ml) was added to 50 ml of Induction medium (0.6% Na₂HPO₄,0.3% KH₂PO₄, 0.05% NaCl, 0.1% NH₄Cl, 0.2% casamino acid, 0.5% glucose, 1mM MgCl₂, 100 μg/ml ampicillin, pH 7.4) and cultured with shaking at 30°C. When OD_(600nm) reached about 0.5, L-tryptophan was added to a finalconcentration of 0.1 mg/ml, followed by culturing with shaking at 30° C.for an additional 6 hours. The culture broth was centrifuged at 3000×gto collect the cells, which were then suspended in 12.5 ml of PBS. Thesuspension was subjected to an ultrasonic generator (Ultrasonics Co.) todisrupt the cells. The disrupt cells were centrifuged at 7000×g for 30minutes to obtain a supernatant liquid as a soluble protein fraction.Ten μl of this soluble protein fraction was subjected to SDSpolyacrylamide (10%) electrophoresis under reducing conditions. As aresult, as shown in FIG. 23, a band with a molecular weight of 40,000which was not detected in the soluble protein fraction of GI724/pTrxFuswas found in the soluble protein fraction of GI724/pTrxhOBM.Accordingly, it was confirmed that a fusion protein (Trx-hOBM) ofthioredoxin and human OBM was expressed in Escherichia coli.

[0314] (3) Binding Capability of Trx-hOBM to OCIF

[0315] Binding of the expressed Trx-hOBM to OCIF was confirmed accordingto the following experiment. Anti-thioredoxin antibody (Invirogen Co.)which was diluted 5000-fold with 10 mM sodium hydrogen carbonatesolution was added to a 96-well immunoplate (Nunc Co.) in the amount of100 μl per well. After being allowed to stand overnight at 4° C., theliquid in the wells was discarded. Two hundred μl of a solution preparedby diluting Block Ace™ (Snow Brand Milk Products Co., Ltd.) two-foldwith PBS (BA-PBS) was added to each well. After being allowed to standfor one hour at room temperature, the wells were washed three times withP-PBS. The soluble protein fractions originating from theabove-described GI724/pTrxhOBM or GI724/pTrxFus, each diluted withBA-PBS in various concentrations were added to each well in the amountof 100 μl. After being allowed to stand for two hours at roomtemperature, each well was washed three times with P-PBS and chargedwith 100 μl of OCIF (100 ng/ml) which was diluted with BA-PBS. Afterbeing allowed to stand for two hours at room temperature, each well waswashed three times with P-PBS and charged with 100 μl ofperoxidase-labeled anti-OCIF antibody (described in WO 96/26217) whichwas diluted 2,000-fold with BA-PBS. After being allowed to stand for twohours at room temperature, each well was washed six times with P-PBS andcharged with 100 μl of TMB solution. After being allowed to stand forabout 10 minutes at room temperature, each well was charged with 100 μlof termination solution (Stopping Reagent). Absorbance of each well at450 nm was measured by a microplate reader. The results are shown inFIG. 24. There was no difference in the absrobance between the samplewith the soluble protein fraction originating from GI724/pTrxFus addedthereto and the sample without the addition of this soluble proteinfraction. On the other hand, the absorbance increased in the samples towhich the soluble protein fraction originating from GI724/pTrxhOBM wasadded in proportion to the concentration of the soluble proteinfraction. The results of the other experiment wherein the dilution rateof the soluble protein fraction was maintained constant (1%) whileadding OCIF diluted with BA-PBS in different concentrations (0-100ng/ml) are shown in FIG. 25. It can be seen that the absorbance remainedlow at any concentrations of OCIF in samples using a soluble proteinfraction originating from GI724/pTrxFus, whereas the absorbanceincreased in proportion to the OCIF concentration in the samples towhich the soluble protein fraction originating from GI724/pTrxhOBM wasadded. Based on these results, it was confirmed that Trx-hOBM which isproduced from GI724/pTrxhOBM has a capability of binding to OCIF.

[0316] (4) Large-Scale Cultivation of Escherichia coli which ProducesTrx-hOBM

[0317] GI724/pTrxhOBM cells were spread on RMG-Amp agar (0.6% Na₂HPO₄,0.3% KH₂PO₄, 0.05% NaCl, 0.1% NH₄Cl, 2% casamino acid, 1.5% agar, pH7.4) using a platinum transfer 100 p. The cells were cultured overnightat 30° C. The cultured cells were suspended in 10 ml of Inductionmedium. The suspension was added (5 ml for each) to two 2 l Erlenmeyerflasks containing 500 ml of Induction medium and cultured at 30° C. withshaking. When the OD_(600nm) reached about 0.5, L-tryptophan was addedto a final concentration of 0.1 mg/ml. Culturing with shaking wascontinued for six hours at 30° C. The culture broth was centrifuged for20 minutes at 3000×g to collect the cells, which were suspended in 160ml of PBS. The suspension was subjected to an ultrasonic generator(Ultrasonics Co.) to disrupt the cells. The supernatant liquid wascentrifuged for 30 minutes at 7000×g to obtain a soluble proteinfraction.

[0318] (5) Preparation of OCIF-Immobilized Affinity Column

[0319] Two g of TSKgel AF-Tolesyl Toyopal 650 (Tosoh Corp.) and 40 ml of1.0 M potassium phosphate buffer (pH 7.5) containing 35.0 mg ofrecombinant OCIF, which was prepared according to the method describedin WO 96/26217, were mixed. The mixture was gently shaken overnight at4° C. to effect a coupling reaction. The reaction mixture wascentrifuged to remove the supernatant. To inactivate excess activeresidues, 40 ml of 0.1 M Tris-HCl buffer (pH 7.5) was added to theprecipitated carrier and the mixture was gently shaken at roomtemperature for one hour. The carrier in a column was washed with 0.1 Mglycine-HCl buffer (pH 3.3) containing 0.01% Polysorbate 80 and 0.2 MNaCl and 0.1 M sodium citrate buffer (pH 2.0) containing 0.01%Polysorbate 80 and 0.2 M NaCl. The carrier in the column wasequilibrated by charging twice with 10 mM sodium phosphate buffer (pH7.4) containing 0.01% Polysorbate 80.

[0320] (6) Purification of Trx-hOBM using OCIF-Immobilized AffinityColumn

[0321] Unless otherwise indicated, purification of Trx-hOBM was carriedout at 4° C. The above-mentioned OCIF-immobilized affinity carrier (10ml) and the soluble protein fraction (120 ml) prepared in Example 25(4)were mixed. The mixture was gently shaken overnight at 4° C. in four 50ml centrifugel tubes using a rotor. An Econo-column™ (internal diameter:1.5 cm, length: 15 cm, manufactured by BioRad Co.) was filled with thecarrier in the mixture. The column was charged with 300 ml of PBScontaining 0.01% Polysorbate 80, 100 ml of 10 mM sodium phosphate buffer(pH 7.0) containing 0.01% Polysorbate 80 and 2.0 M NaCl, and 100 ml of0.1 M glycine-HCl buffer (pH 3.3) containing 0.01% Polysorbate8 0 and0.2 M NaCl, in that order. Next, proteins adsorbed in the column wereeluted with 0.1 M sodium citrate buffer (pH 2.0) containing 0.01%Polysorbate 80 and 0.2 M NaCl. The eluate was collected in 5 mlportions. Each fraction thus collected was immediately neutralized withaddition of 10% volume of 2 M Tris buffer solution (pH 8.0). Presence orabsence of Trx-hOBM in the eluted fractions was determined according tothe method previously described in Example 25(3) (the binding capabilityto OCIF). The fractions containing Trx-hOBM were collected and purifiedfurther.

[0322] (7) Purification of Trx-hOBM by Gel Filtration

[0323] About 25 ml of Trx-hOBM fractions obtained in Example 25(6) wasconcentrated to about 0.5 ml by centrifuging using Centriplus 10 andCentricon 10 (Amicon Co.). This sample was applied to a Superose 12 HR10/30 column (1.0×30 cm, Pharmacia Co.) previously equilibrated with PBScontaining 0.01% Polysorbate 80. For the separation, PBS containing0.01% Polysorbate 80 was used as a mobile phase at a flow rate of 0.25ml/min. The eluate from the column was collected in 0.25 ml portions.The Trx-hOBM in the thus-collected fractions was detected by the samemethod as previously described in the Example 25(3) and SDS-PAGE.Fractions containing purified Trx-hOBM were collected and the proteinconcentration of Trx-hOBM was determined. The measurement of the proteinconcentration was carried out using bovine serum albumin as a standardsubstance using DC-Protein assay kit (BioRad Co.).

Example 26

[0324] Osteoclast Formation-Inducing Activity of hOBM

[0325] phOBM and pcDL-SR α296 were respectively transfected into COS-7cells using Lipofectamine (Gibco Co.). The cells were cultured for oneday in DMEM containing 10% FCS, trypsinized, plated on cover slips (15mm round shape, manufactured by Matsunami Co.) in 24-well plates at5×10⁴ cells per well, and cultured for 2 days. The culture plate waswashed once with PBS. The cells were fixed with PBS containing 1%paraformaldehyde at room temperature for 8 minutes. The plate on whichthe fixed cells were attached was washed 6 times with PBS, then 700 μlof mouse spleen cells suspended at 1×10⁶/ml in α-MEM containing 10⁻⁶ Mactive-form vitamin D₃, 10⁻⁷ M dexamethasone, and 10% fetal bovine serumwere added to each well. Millicell PCF (Millipore Co.) was set in eachwell and a suspension of ST2 cells in the above-mentioned culture medium(4×10⁴/ml) were added, 700 μl per well, into the Millicell PCF followedby incubation at 37° C. for 6 days. After the culture, the Millicell PCFwas removed, the plate was washed once with PBS, and the cells werefixed with acetone-ethanol solution (50:50) for one minute. Then, thecells exhibiting tartaric acid-resistant acid phophatase activity(TRAP), which is a specific marker for osteoclast, were selectivelystained using LEUKOCYTE ACID PHOSPHATASE kit (Sigma Co.). As a result ofmicroscopic observation, TRAP-positive cells were not detected in thewells in which COS-7 cells transfected with pcDL-SR α296 were fixed. Incontrast, 65±18 (average±standard deviation, n=3) TRAP positive cellswere observed in the wells in which COS-7 cells transfected with phOBMwere fixed. Moreover, expression of calcitonin receptor was confirmed bythe fact that ¹²⁵I-labeled salmon calcitonin (Amersham Co.) specificallybound to these TRAP positive cells. Based on these findings, it has beenproven that human OBM, which is the protein encoded by cDNA of thepresent invention, has osteoclast formation-inducing activity.

Example 27

[0326] Osteoclast Formation-Inducing Activity of Trx-hOBM andSecreted-Form Human OBM

[0327] Mouse spleen cells were suspended in α-MEM containing 10⁻⁸ Mactive-form vitamin D₃, 10⁻⁷ M dexamethasone, and 10% fetal bovine serumat a concentration of 2×10⁶/ml. The suspension was added to a 24 wellplate in the amount of 350 μl per well. Each well was then charged with350 μl of a solution prepared by diluting purified Trx-hOBM with theabove-mentioned culture medium (40 ng/ml), 350 μl of solution preparedby 10-fold diluting a conditioned medium which was produced by culturing293-EBNA cells, onto which pCEPshOBM or pCEP4 were transfected, in aculture medium IMDM-10% FCS, with above-mentioned culture medium, or 350μl only of the above-mentioned culture medium. The Millicell PCF(Mollipore Co.) was placed on each well, to which 600 μl of ST2 cellswhich were suspended in the above-mentioned culture medium (4×10⁴/ml)were added. After cultured for six days, the Millicell PCF was removed.The plate was washed once with PBS and the cells were fixed byacetone-ethanol solution (50:50) for one minute. Then, the cellsexhibiting the activity of tartaric acid resistant acidic phophatase(TRAP activity) were selectively stained using LEUKOCYTE ACIDPHOSPHATASE kit (Sigma Co.). The results of microscopic observationrevealed that no cells exhibiting the TRAP activity were detected in thewells to which no Trx-hOBM was added, whereas 115±19 (average±standarddeviation, n=3) TRAP-positive cells were observed in the wells to whichTrx-hOBM was added. Similarly, while no cells exhibiting TRAP activitywere detected in the wells to which conditioned medium of 293-EBNA cellstransfected with pCEP4 had been added, 125±23 (average±standarddeviation, n=3) TRAP positive cells were observed in the wells to whichconditioned medium of 293-EBNA cells transfected with pCEPshOBM had beenadded. Moreover, expression of calcitonin receptor was confirmed by thefact that ¹²⁵I-labeled salmon calcitonin (Amersham Co.) specificallybinds to these TRAP positive cells. These results have proven thatTrx-hOBM and secreted-form hOBM exhibit osteoclast formation-inducingactivity.

Example 28

[0328] Preparation of Polyclonal Antibody

[0329] Mouse sOBM or human sOBM used as an immunogen was preparedaccording to the method described above. Especially, mouse sOBM cDNA(cDNA (Sequence ID No. 18) encoding mouse sOBM (Sequence ID No. 16)which does not have the membrane binding region of the mouse OBM due toabsence of the amino acids from the N-terminal down to the 72nd aminoacid) or human sOBM cDNA (cDNA (Sequence ID No. 19) encoding human sOBM(Sequence ID No. 17) which does not have the membrane binding region ofhuman OBM due to absence of the amino acids from the N-terminal down tothe 71st amino acid) was ligated with a Hind III/EcoRV fragment (5.2 kb)of the expression vector psec TagA (InVitrogen Co.) including thenucleotide sequence encoding a signal peptide of κ-chain ofimmunoglobulin, together with an EcoRI/PmaCl fragment (0.32 kb) of OBMcDNA, using a ligation kit ver. 2 (Takara Shuzo Co.). Escherichia coliDH5 α was transformed with the reaction product. The plasmids obtainedfrom the resulting ampicillin resistant strains were purified by thealkali SDS and digested with an restriction enzyme to select a plasmidwith 0.6 Kb and 0.32 kb fragments inserted into psec TagA. The sequenceof this plasmid was identified using the Dyedeoxyterminator CycleSequencing FS kit (product of Perkin Elmer Co.). As a result, it wasconfirmed that this plasmid has a sequence encoding mouse or human sOBM.After plasmid was digested with restriction enzymes NheI/XhoI, afragment (1.0 kb) corresponding to secretion form OBM cDNA was recoveredby agarose gel electrophoresis. This fragment was inserted into anNheI/XhoI fragment (10.4 kb) of the expression vector pCEP4 (InVitrogenCo.) using a ligation kit. Escherichia coli DH5 α was transformed usingthe reaction product. Plasmids were purified by the alkali SDS from theresulting ampicillin resistant strains. Analyzing these plasmids bydigesting with a restriction enzyme, Escherichia coli possessing asecretion type OBM expression plasmid (pCEP sOBM) having the objectivestructure was selected. The Escherichia coli strain having the pCEP sOBMwas cultured and pCEP sOBM was purified using a Qiafilter plasmid midykit (Qiagen Co.). Next, 293-EBNA cells were suspended in IMDM (IMDM-10%FCS) containing 10% FCS and plated onto a 24-well plate coated withcollagen (product of Sumitomo Bakelite Co., Ltd.) at a cell density of2×10⁵ cells/2 ml/well. After culturing overnight, the cells weretranformed with 1 μg of pCEP sOBM or pCEP4 using 4 μl of Lipofectamine(Gibco Co.) and further cultured for two days in 0.5 ml of serum-freeIMDM or IMDM-10% FCS. The culture supernatant was recovered. A cell linewith high productivity of recombinant mouse soluble OBM (msOBM) or humansoluble OBM (hsOBM) was screened as follows. Sodium bicarbonate wasadded to the culture supernatant which is assumed to contain msOBM orhsOBM to a final concentration of 0.1 M. One hundred μl of the culturesupernatant was added to each well in 96-well immunoplates (Nunc Co.)and allowed to stand overnight at 4° C., thereby msOBM or hsOBM in theculture supernatant was immobilized on each well. To each well, 200 μlof Block Ace™ (Snow Brand Milk Products Co., Ltd.) solution dilutedfour-fold with PBS (B-PBS) was added and the plates were allowed tostand for two hours at room temperature. After washing each well in theplates three times with PBS (P-PBS) containing 0.1% Polysorbate 20, 100μl of each recombinant OCIF (rOCIF) solution (3-100 ng/ml) dilutedserially with P-PBS was added to each well in the plates. The plateswere allowed to stand for two hours at 37° C. After washing the platesthree times with PBS, 100 μl of a peroxidase-labeled anti-OCIFpolyclonal antibody (WO 96/26217) diluted with B-PBS was added to eachwell. After allowing to stand for two hours at 37° C., the wells werewashed six times with P-PBS. Then, 100 μl of TMB solution (TMB SolubleReagent, High Sensitivity, ScyTek Co.) was added to each well in theplates and the plates were allowed to stand at room temperature forabout 10 minutes, subsequently the reaction was terminated by adding 100μl of a stopping solution (Stopping Reagent, ScyTek Co.) to each well.Absorbance at 450 nm of each well was measured using a microplatereader. It was confirmed that the absorbance increased remarkably inproportion to concentration of the added OCIF in the plates in whichmsOBM or hsOBM in the culture supernatant of the cell line producingmsOBM or hsOBM was immobilized therein.

[0330] The cell line that exhibited a high rate of increase inabsorbance was selected as a a strain with high productivity.Thus-related 293-EBNA cells with high productivity of msOBM or hsOBMwere cultured on a large scale in an IMDM medium containing 5% FCS,using 25 T-flasks (T-225) . After the cell reached to confluent, a freshculture medium was added to each T-225 flask in the amount of 100 ml perflask and cells were cultured for 3-4 days, to collect the culturesupernatant. These procedures were repeated four times to obtain 10 L ofthe culture supernatant containing msOBM or hsOBM. Purified msOBM (10mg) or hsOBM (12 mg), which shows homogeneous band (molecular weight: 32kDa) on SDS-polyacrylamide gel electrophoresis, were obtained from theculture supernatant by means of affinity chromatography on anOCIF-immobilized column and gel filtration chromatography according tothe method described in examples 25(6) and 25(7). Each thus-obtainedpurified preparation was used as an antigen for immunization. Eachprotein antigen obtained was dissolved in phosphate buffered saline(PBS) to a concentration of 200 μg/ml and emulsified with an equivalentvolume of Freund's complete adjuvant. One ml of the emulsion wassubcutaneously immunized to each of three Japanese white rabbits aboutonce every week. A booster injection was given when the antibody titerreached a peak. Whole blood was collected 10 days thereafter. The serumwas diluted two-fold with a binding buffer for protein A sepharosechromatography (BioRad Co.) and applied to a protein A columnequilibrated with the same buffer. After washing the column extensivelywith the same buffer, the anti-sOBM antibody adsorbed to the column waseluted with an elution buffer (BioRad Co.) or 0.1 M glycine-HCl buffer,pH 3.0. To neutralize the eluate immediately, the eluate wasfractionated using test tubes containing a small amount of 1.0 MTris-HCl (pH 8.0). The eluate was dialyzed against PBS overnight at 4°C. The antibody content in the antibody solution was measured by theLowry method using bovine IgG as a standard protein. Thus, about 10 mgof purified immunoglobulin (IgG) containing the polyclonal antibody ofthe present invention per 1 ml of rabbit antiserum was obtained.

Example 29

[0331] Measurement of OBM and sOBM by ELISA Using Polyclonal Antibody

[0332] A sandwich ELISA was constructed using the rabbit anti-human sOBMpolyclonal antibody obtained in Example 28 as the solid phase antibodyand enzyme-labeled antibody. Peroxidase (POD)-labeled antibody wasprepared according to the method of Ishikawa (lshikawa et al., J.Imunoassay, Vol. 4, 209-327, 1983).

[0333] The anti-human sOBM polyclonal antibody obtained in the Example28 was dissolved in a 0.1 M NaHCO₃ to a concentration of 2 μg/ml. Onehundred μ l of the resulting solution was added to each well in 96-wellimmunoplates (Nunc Co.), which was then allowed to stand at roomtemperature overnight. Next, 200 μl of 50% Block Ace™ (Snow Brand MilkCo., Ltd.) was added to each well and the plates were allowed to standfor one hour at room temperature. The wells were washed three times withPBS containing 0.1% Polysorbate 20 (washing buffer).

[0334] Human OBM was expressed according to the method of Example 26 andpurified according to the method of Example 2. The purified human OBMand the purified human sOBM prepared in example 28 were serially dilutedwith the first reaction buffer (0.2 M Tris-HCl buffer, pH 7.2,containing 40% Block Ace and 0.1% Polysorbate 20) , respectively, and100 μl of the diluted solution was added to each well in the plates. Theplates were allowed to stand at room temperature for two hours, andwashed three times with the above-mentioned washing buffer.Subsequently, 100 μl of POD-labeled anti-human sOBM polyclonal antibodydiluted 1000-fold with the second reaction buffer (0.1 M Tris-HClbuffer, pH 7.2, containing 25% Block Ace and 0.1% Polysorbate 20) wasadded to each well in the plates. After the plates were allowed to standat room temperature for two hours, each well was washed three times withthe washing buffer. Next, 100 μl of enzyme substrate solution (TMB,ScyTek Co.) was added to each well in the plates, and the plates wereallowed to stand for 10 minutes, followed by the addition of 100 ml of areaction termination solution (Stopping reagent, ScyTek Co.) to stop theenzyme reaction. The absorbance at 450 m of each well was measured usinga microplate reader. The results are shown in FIG. 26. The sandwichELISA using a rabbit anti-human sOBM polyclonal antibody recognizedalmost equally human sOBM (molecular weight, about 32 kDa) and human OBM(molecular weight, about 40 kDa), with a measurement sensitivity ofabout 12.5×10⁻³ pmol/ml (human OBM: about 500 pg/ml, human sOBM: about400 pg/ml). The measurement of mouse sOBM and mouse OBM by ELISA usingthe rabbit anti-mouse sOBM polyclonal antibody obtained in the Example28 was able to be carried out in the same manner. It was confirmed thatan extremely small amount of mouse sOBM or mouse OBM can be measuredwith almost the same sensitivity as described above.

[0335] As mentioned above, the anti-human sOBM polyclonal antibody ofthe present invention prepared in the Example 28 can equally recognizeboth the human sOBM and human OBM antigens. Therefore, the antibody wasnamed anti-human OBM/sOBM polyclonal antibody. Similarly, the anti-mousesOBM polyclonal antibody prepared in the Example 28 can equallyrecognize both the mouse sOBM and mouse OBM antigens. This antibody wastherefore named anti-mouse OBM/sOBM polyclonal antibody.

Example 30

[0336] Preparation of Monoclonal Antibody

[0337] The purified human sOBM prepared in the Example 28 was used asthe antigen for immunization. The purified human sOBM was dissolved inphysiological saline solution to a concentration of 10 μg/ml andemulsified by mixing with an equivalent volume of Freund's completeadjuvant. The emulsion was intraperitoneally administered to BALB/c miceat a dose of 200 μl three times, once a week, to immunize mice. Next,the equivalent volume of the Freund's complete adjuvant was added to aphysiological saline solution containing 5 μg/ml of human sOBM and themixture was sufficiently emulsified. This emulsion was injectedintraperitoneally to BALB/c mice at a dose of 200 μl, once a week forfour weeks for immunization. One week after the fourth immunization, 100μl of a physiological saline solution containing 10 μg/ml of human sOBMwas intravenously administered to the BALB/c mice as a booster. Afterthree days, the spleen was extracted and spleen cells were separated.The spleen cells were fused with mouse myeloma cells, P3×63-Ag8.653according to a conventional method (Koehler, G. and Milstein, C.,Nature, 256, 495 (1975)) The suspended fused cells were cultured for 10days in an HAT medium containing hypoxanthine, aminopterin, andthymidine. After the myeloma cells were dead-and hybridomas appeared,the HAT medium was replaced with an aminopterin-free HAT medium, and thecell culture was continued.

Example 31

[0338] Selection of Hybridoma and Cloning

[0339] Appearance of hybridomas was recognized 10 days after cell fusionin Example 30. Monoclonal antibodies recognizing the human sOBM withhigh affinity and hybridomas producing these antibodies were selectedaccording to the following procedure using the improved solid phaseELISA which is described below. In addition, to select the anti-OBMmonoclonal antibody which recognizes both human sOBM and mouse sOBM,mouse sOBM prepared in the Example 27 was used in addition to human sOBMas the antigen for the solid phase ELISA. The human sOBM and mouse sOBMwere respectively dissolved in a 0.1 M sodium bicarbonate solution at aconcentration of 5 μg/ml. Fifty ml of each antigen solution was added toeach well in 96-well immunoplates. (Nunc Co.). The plates were allowedto stand at 4° C. overnight to immobilize the antigens. The antigensolution in each well was discarded. Each well was then filled with 200μl of 50% Block Ace™ (Snow Brand Milk Products Co., Ltd.) and allowed tostand at room temperature for one hour. After each well was washed withphosphate buffered saline solution (PBS-P) containing 0.1% Polysorbate20 , 40 μl of calf serum (Hiclone Inc.) was added to each well.Subsequently, 10 μl of each hybridoma culture supernatant was added toeach well and each well was incubated at room temperature for two hoursin the presence of 80% calf serum. The object of the solid phase ELISAin the presence of 80% calf serum is to select a hybridoma which producean antibody which can detect a very small amount of human sOBM or mousesOBM even in a solution containing high concentration of protein and inthe presence of an immunoreaction interfering substance derived fromserum, i.e. a hybridoma which can produce an antibody with a highaffinity for human sOBM or mouse sOBM. After the reaction at roomtemperature for two hours, the plates were washed with PBS-P andsubsequently, 50 μl of peroxidase-labeled anti-mouse IgG (KPL Co.)diluted 5000-fold with physiological saline solution containing 25%Block Ace was added to each well. After the reaction at room temperaturefor two hours, the plate was washed three times with PBS-P. After theaddition of 50 μl of an enzyme substrate solution (TMB, ScyTek Co.) toeach well, the reaction was continued at room temperature for fiveminutes. The enzymatic reaction was stopped by the addition of 50 μl ofa termination solution (stopping reagent, ScyTek Co.). Hybridomas whichproduce antibodies recognizing human sOBM or mouse sOBM were selected bymeasuring absorbance at 450 nm of each well using a microplate reader(Immune Reader NJ2000™, Nippon InterMed Co.). Hybridomas producingantibodies exhibiting particularly high absorbance (OD_(450nm)) wereselected Cloning of these hybridomas by a limiting dilution method wasrepeated 3 to 5 times to establish stable hybridomas. Hybridomasexhibiting particularly high antibody productivity were selected amongthe established antibody-producing hybridoma clones.

Example 32

[0340] Production and Purification of Monoclonal Antibody

[0341] The antibody-producing hybridomas obtained in the Example 31,i.e. high affinity antibody-producing hybridoma which recognizes humansOBM and hybridoma which produces an antibody showing cross-reactivityto the mouse sOBM were cultured, respectively. Each hybridoma wasimplanted intraperitoneally to BALB/c mice (1×10⁶ cells per mouse) towhich pristan (Aldorich Co.) was administered one week previously. Afterabout 2-3 weeks, accumulated ascites were collected. The monoclonalantibody, which recognizes human sOBM of the present invention or boththe human sOBM and mouse sOBM in the ascites, was purified according tothe purification method of anti-OBM/sOBM polyclonal antibodies using aProtein A column described in the Example 28. The purified monoclonalantibody was thus obtained from the ascites by Protein A columnchromatography (Pharmacia Co.).

Example 33

[0342] Antigen Specificity of Monoclonal Antibody

[0343] The antigen specificity of a monoclonal antibody whichspecifically recognizes human sOBM and the monoclonal antibodyexhibiting cross-reactivity to both the human sOBM and mouse sOBM wasinvestigated using human sOBM, human intact OBM having a membranebinding region, mouse sOBM, and mouse intact sOBM having a membranebinding region. More than thirty kinds of monoclonal antibody wereobtained. The results on several representative antibodies are shown inTable 1. As a result, it was found that most anti-human sOBM monoclonalantibodies which specifically recognize human sOBM also recognize thehuman intact OBM having a membrane binding region, but not the mouse OBMand the mouse intact OBM which has a membrane binding region. On theother hand, it was found that only a few monoclonal antibodiesrecognizing both the human sOBM and mouse sOBM were obtained and thatthese antibodies exhibit cross-reactivity to both the human OBM andmouse OBM. These results show that there is a common antigen-recognizingsites, namely a common epitopes, in both the human OBM and mouse OBM.Based on the fact that the anti-human sOBM monoclonal antibody preparedusing the human sOBM as an immune antigen also equally recognizes humanOBM having a membrane binding region. Anti-human sOBM monoclonalantibody was named the anti-human OBM/sOBM monoclonal antibody. TABLE 1Antigen Antibody hsOBM hOBM MsOBM mOBM H-OBM 1 + + − − H-OBM 2 + + − −H-OBM 3 + + − − H-OBM 4 + + − − H-OBM 5 + + − − H-OBM 6 + + − − H-OBM7 + + − − H-OBM 8 + + − − H-OBM 9 + + + + H-OBM 10 + + − − H-OBM 11 + +− − H-OBM 12 + + − − H-OBM 13 + + + + H-OBM 14 + + − −

Example 34

[0344] Determination of Class and Subclass of Monoclonal Antibody

[0345] The class and subclass of the monoclonal antibody of the presentinvention were determined by the immunoglobulin class and subclassanalysis kit (Amersham Co.) according to the protocol indicated. Theresults on representative monoclonal antibodies are shown in Table 2. Asshown in Table 2, the majority of anti-human OBM/sOBM monoclonalantibodies were IgG₁, the others were IgG_(2a) and IgG_(2b). Lightchains for all antibodies were κ chains. TABLE 2 Antibody IgG₁ IgG_(2a)IgG_(2b) IgG₃ IgA κ H-OBM 8 − + − − − + H-OBM 9 + − − − − + H-OBM 10 + −− − − + H-OBM 11 + − − − − + H-OBM 12 − − + − − + H-OBM 13 + − − − − +H-OBM 14 + − − − − +

Example 35

[0346] Measurement of the Dissociation Constant (K_(d) Value) ofMonoclonal Antibody

[0347] The dissociation constant of the monoclonal antibody was measuredaccording to a known method (Betrand Friguet et al.: Journal ofImmunological Methods, 77, 305-319, 1986). That is, the purifiedantibody obtained in the Example 32 was diluted with 0.4 M Tris-HClbuffer (a primary buffer, pH 7.4) containing 40% Block Ace and 0.1%Polysorbate 20 to give a concentration of 5 ng/ml. The solution wasmixed with an equivalent volume of a diluted solution of purified humansoluble OBM (hsOBM) obtained in Example 28 in the primary buffer with aconcentration range of 6.25 to 10 μg/ml. The mixture was allowed tostand for 15 hours at 4° C. to bind the hsOBM to the monoclonalantibody. After 15 hours, the antibody not bound to the hsOBM (10 μg/ml,100 μl/well) was measured using an immobilized solid phase ELISA tocalculate the dissociation constant of the monoclonal antibody to thehsOBM. In addition, affinity to msOBM of an antibody, which is amonoclonal antibody for the hsOBM and also exhibits the cross-reactivityto mouse soluble OBM (msOBM), was measured according to the same methodexcept for using msOBM instead of the hsOBM. Dissociation constant ofantibodies, which exhibit high affinity to each antigen and are usefulfor enzymatic immunoassay and binding assay, are shown in Table 3. TABLE3 Antibody Subclass Antigen Dissociation constant Kd(M) H-OBM 1 IgG₁(κ)hsOBM 1 × 10⁻¹¹ < kd < 1 × 10⁻¹⁰ H-OBM 4 IgG₁(κ) hsOBM 1 × 10⁻¹¹ < kd <1 × 10⁻¹⁰ H-OBM 9 IgG₁(κ) hsOBM 1 × 10⁻⁹ < kd < 1 × 10⁻⁸ H-OBM 9 IgG₁(κ)msOBM 1 × 10⁻⁸ < kd < 1 × 10⁻⁷

[0348] As a result, the dissociation constants (Kd) of H-OBM 1 and H-OBM4 which are the antibodies specific to human soluble OBM (hsOBM) were inthe order of 10⁻¹¹ M, indicating the high affinity to hsOBM. On theother hand, the Kd value of the antibody H-OBM 9 which recognizes boththe hsOBM and mouse soluble OBM (msOBM) was in the order of 10⁻⁸ M tomsOBM and in the order of 10⁻⁹ M to hsOBM. In addition, the dissociationconstant of the other antibody which recognizes both antigens in theTable 1, i.e. the dissociation constant of H-OBM 13 for each antigen,was the same as that of H-OBM 9, and these two antibodies belong to thesame subclass. These findings suggest the possibility that they are theidentical antibodies which recognize the same epitope of each antigen.

Example 36

[0349] Measuring Method of Human OBM and sOBM by Sandwich ELISA usingAnti-Human OBM/sOBM Monoclonal Antibodies

[0350] A sandwich ELISA was constructed using the two high affinitymonoclonal antibodies obtained in Example 35, H-OBM 1 and H-OBM 4,respectively as a solid phase antibody and an enzyme-labeled antibody.Labeling of the antibody was carried out using a maleimideactivated-peroxidase kit (Piers Co.). The antibody, H-OBM 1, wasdissolved in a 0.1 M sodium bicarbonate solution to a concentration of10 μg/ml, and 100 μl of the solution was added to each well in 96-wellimmunoplates (Nunc company). After being allowed to stand overnight at4° C. to immobilize the antibody, the solution was discarded and 300 μ lof 50% Block Ace™ solution was added to each well in the plates. Eachwell in the plates was blocked by allowing to stand at room temperaturefor two hours. After blocking, the plates were washed with phosphatebuffered saline containing 0.1% Polysorbate 20 (PBS-P). Human OBM (hOBM)and human soluble OBM (hsOBM) were respectively diluted with 0.4 MTris-HCl buffer, pH 7.4, containing 40% Block Ace™ (Snow Brand MilkProducts Co., Ltd.) and 0.1% Polysorbate 20 (Wako Pure Chemicals Co.,Ltd.) (the first reaction buffer) to prepare test samples with variousconcentrations. These test samples with different concentrations wereadded to each well in the amount of 100 μl per well and reacted to theantibody, H-OBM 1 immobilized on each well by incubating at roomtemperature for two hours. After two hours, the plates were washed withPBS-P. Next, 100 μl of a solution of POD-labeled H-OBM 4 antibody in 0.2M Tris-HCl buffer, pH 7.4, containing 25% Block Acem and 0.1%Polysorbate 20 (the second reaction buffer) was added to each well,followed by further incubating at room temperature for two hours. Theplates were then washed with PBS-P and 100 μl of an enzyme substratesolution (TMB, ScyTek Co.) was added to each well to start enzymereaction. The enzyme reaction was terminated by the addition of 100 μlof a reaction termination solution (stopping reagent, ScyTek Co.) toeach well. The absorbance of each well at 450 nm was measured using amicroplate reader. The results are shown in FIG. 27.

[0351] As a result, it was confirmed that the sandwich ELISA constructedusing the two anti-human OBM/sOBM monoclonal antibodies, H-OBM 1 andH-OBM 4 with high affinity for human OBM/sOBM prepared in the Example35, equally recognizes human OBM and human sOBM, and is able to measurea very small amount of human OBM or human sOBM at a quantitative limitof about 1.25×10⁻³ to 2.5×10⁻³ pmol/ml (about 50-100 pg/ml for human OBMwith a molecular weight of 40 kDa, about 40-80 pg/ml for human sOBM witha molecular weight of 32 kDa). The hybridomas which produce these twoanti-human OBM/sOBM monoclonal antibodies, H-OBM 1 and H-OBM 4 werenamed H-OBM1 and H-OBM4, respectively. The hybridoma producinganti-human OBM/sOBM monoclonal antibody (H-OBM 9) which recognizes mouseOBM and mouse sOBM and also has an osteoclast formation-inhibitoryactivity was named H-OBM9. These hybridomas were deposited with theNational Institute of Bioscience and Human Technology, the Agency ofIndustrial Science and Technology, on Nov. 5, 1993 with Deposition Nos.FERM BP-6264 (H-OBM1), FERM BP-6265 (H-OBM 4), and FERM BP-6266 (H-OBM9).

Example 37

[0352] Measurement of Mouse OBM and Mouse sOBM using Anti-Human OBM/sOBMMonoclonal Antibodiy which Recognizes Mouse OBM and Mouse sOBM

[0353] A sandwich ELISA was constructed using the anti-human OBM/sOBMmonoclonal antibody, H-OBM 9, which recognizes mouse OBM and mouse sOBMobtained as an solid phase antibody in the Examples 33 and 35, and theanti-mouse OBM/sOBM polyclonal antibody as an enzyme-labeled antibodyobtained in the example 28. The mouse OBM and mouse sOBM wererespectively diluted with the first reaction buffer to give variousconcentrations in the same manner as in the Example 35 and then measuredsOBM according to the method described in the Example 36. The resultsare shown in FIG. 28. As a result, it was found that mouse OBM and mousesOBM can be similarly measured using H-OBM 9 which is the anti-humanOBM/sOBM monoclonal antibody recognizing the mouse OBM and mouse sOBM ofthe present invention. As shown by the result of Example 35, thisanti-human OBM/sOBM monoclonal antibody H-OBM 9 has a high dissociationconstant relative to the mouse sOBM, namely it has a comparatively lowaffinity to mouse sOBM. The sensitivity in the measurement of mouse OBM(molecular weight, about 40 kDa) and mouse sOBM (molecular weight, about32 kDa) by this ELISA assay was about 25×10⁻³ pmol/ml (about 1 ng/ml formouse OBM and about 0.8 ng/ml for mouse sOBM).

Example 38

[0354] Osteoclastogenesis-Inhibitory Activity of Anti-OBM/sOBM Antibody

[0355] It is known that osteoclast-like cells (OCL) are induced byco-culture of mouse spleen cells and ST2 cells (mouse bonemarrow-derived stromal cells; Endocrinology, 125, 1805-1813 (1989)).Capability of the anti-OBM/sOBM antibody to inhibit the OCL formationwhen added to the co-culture system was studied. Because the mouse OBMis expressed in this co-culture system, a rabbit anti-mouse OBM/sOBMpolyclonal antibody which recognizes mouse OBM and an anti-humanOBM/sOBM monoclonal antibody (H-OBM 9) which recognizes both human OBMand mouse OBM antigens were used as the antibodies in this example.Seven hundred microliters per well of each anti-OBM antibody dilutedserially with α-MEM containing 10% FCS and 350 μl/well of male mousesplenocytes (2×10⁶/ml)suspended in the same medium described above wereadded to each well in a 24-well plate (Nunc). Next, ST2 cellstrypsinized and suspended in the above-mentioned culture mediumcontaining 4×10⁻⁸ M Vitamin D₃ and 4×10⁻⁷ M Dexamethazone (8×10⁴cells/ml) were added to each well in the amount of 350 μl/well, followedby culturing for six days at 37° C. After the plates were washed oncewith PBS, cells in each well were fixed with a mixture of ethanol andacetone (50:50) for one hour at room temperature. The plates were driedin air, and 500 μl of substrate solution was added to each wellaccording to the protocol of the LEUKOCYTE ACID PHOSPHATASE kit (SigmaCo.), followed by incubating for 55 minutes at 37° C. Only the cellsexhibiting the tartaric acid-resistant acid phophatase activity (TRAPactivity), which is a specific marker for osteoclasts, were stained bythis reaction. The plates were washed once with distilled water anddried in air, and the number of TRAP-positive cells was counted. Theresults are shown in Table 4. As shown in Table 4, both the rabbitanti-mouse OBM/sOBM polyclonal antibody and the anti-human OBM/sOBMmonoclonal antibody, H-OBM 9, which recognizes mouse OBM inhibited OCLformation in a dose-dependent manner. These antibodies were found topossess osteoclastogenesis-inhibitory activity likeosteoclastogenesis-inhibitory factor, OCIF/OPG, and thus are promisingas a therapeutic agent for treating bone metabolism abnormalitysymptoms. TABLE 4 Number of TRAP-positive multinucleates Amount ofRabbit anti-mouse antibody OBM/sOBM Mouse anti-human OBM/sOBM (μg/ml)polyclonal antibody monoclonal antibody (H-OBM 9) 0 1155 ± 53  1050 ±45  10 510 ± 24 650 ± 25 100 10 ± 3 15 ± 4

Example 39

[0356] Human Osteoclast Formation-Inducing Activity of Trx-OBM

[0357] Mononuclear cells were prepared from whole blood collected fromthe vein of a healthy adult by density gradient using Histopaque (SigmaCo.) according to the protocol attached thereto. The mononuclear cellswere suspended at a cell density of 1.3×10⁶/ml in α-MEM containing 10⁻⁷M Dexamethasone, 200 ng/ml macrophage colony stimulating factor (TheGreen Cross Corp.), 10% fetal bovine serum, and purified Trx-OBM (0-100ng/ml) obtained in Example 15. The cell suspension was added to eachwell in 48-well plates in the amount of 300 μl per well, and the cellswere cultured at 37° C. for three days. After the culture broth wasreplaced with the above-mentioned culture medium, the cells werecultured at 37° C. for four days. The cultured cells having tartaricacid resistant acid phosphatase activity (TRAP activity) wereselectively stained according to the method described in Example 5. Thenumber of stained multinucleates was measured by microscope observation.The results are shown in FIG. 29. It was confirmed that TRAP-positivemultinucleates were induced in a dose dependent manner by addition ofTrx-OBM, while no TRAP-positive cells were detected in the wells towhich Trx-OBM was not added. Moreover, these TRAP-positivemultinucleates were found positive to vitronectin receptor which is amarker for osteoclasts. Furthermore, when similar cell culture wascarried out on ivory slices placed on each well in a 48-well plate, pitformation was observed on the ivory slices only in the presence ofTrx-OBM. Based on these findings, Trx-OBM was formed to have theactivity of inducing human osteoclast formation.

Example 40

[0358] Inhibition of Bone Resorbing Activity by Anti-OBM/sOBM Antibody

[0359] [⁴⁵Ca]—CaCl₂ solution (Amersham Co.) was subcutaneously injectedto ddY mouse (Japan SLC Co.) in the 15th day of pregnancy at a dose of25 μCi per mouse to label the bone of the fetus with ⁴⁵Ca. Next day, themouse was sacrificed to obtain the fetus. The forefoot of the fetus wasdrawn and the skin and muscle were removed to obtain the long bones. Thecartilage was removed to obtain the shafts of long bones. The shafts oflong bones were floated one by one in 0.5 ml of culture medium (BGJbmedium (GIBCO BRL company) containing a 0.2% bovine serum albumin (SigmaCo.)) in each well in 24-well plates, and cultured for 24 hours at 37°C. in 5% CO₂. After the pre-cultivation, the bones were transferred intovarious fresh culture media (0.5 ml), each containing one of differentbone resorbing factors (vitamin D₃, prostaglandins E₂, parathyroidhormone, interleukin 1α), and normal rabbit IgG (100 μg/ml; as acontrol), or the rabbit anti-OBM/sOBM polyclonal antibody prepared inExample 28, followed by further cultivation for 72 hours. After thecultivation, the long bones were placed in 0.5 ml of an aqueous solutionof 5% trichloroacetic acid (Wako Pure Chemicals Co., Ltd.), and allowedto stand at room temperature for more than 3 hours to decalcify. Five mlof a scintillator (AQUASOL-2, PACKARD Co.) was added to the culturebroth and the extract of the trichloroacetic acid solution (each 0.5 ml)to measure the radioactivity of ⁴⁵Ca, whereby the ratio of the ⁴⁵Cawhich was liberated into the culture broth by bone resorption wascalculated. The results are shown in FIGS. 30 to 33. As a result,vitamin D₃ (10⁻⁸ M) was found to increase the bone resorbing activity,but the rabbit anti-OBM/sOBM polyclonal antibody suppressed the boneresorption stimulated by vitamin D₃ in a concentration-dependent manner,completely inhibiting the increased bone resorption at a concentrationof 100 μg/ml (FIG. 30). Prostaglandins E₂ (10⁻⁶ M) and parathyroidhormone (100 ng/ml) also increased the bone resorbing activity. However,addition of 100 μg/ml of the rabbit anti-OBM/sOBM polyclonal antibodyalmost completely inhibited the bone resorption stimulated byprostaglandins E₂ and parathyroid hormone (FIGS. 31 and 32). On theother hand, normal rabbit IgG (100 μg/ml), which was used as a positivecontrol, did not affect the bone resorbing activity induced byprostaglandins E₂and parathyroid hormone. Bone resorption was alsoincreased by interleukin 1α (10 ng/ml),but significantly inhibited bythe addition of rabbit anti-OBM/sOBM polyclonal antibody (100 μg/ml)(FIG. 23) Based on these results, it is clear that the antibody of thepresent invention is a superior substance as a bone resorptioninhibitor. The results obtained by similar experiment using H-OBM 9which is a mouse anti-human OBM/sOBM antibody, confirmed that thisantibody exhibits an almost equivalent bone resorption-inhibitory effectas the rabbit anti-OBM/sOBM polyclonal antibody.

[0360] Industrial Applicability

[0361] The present invention provides a novel protein that specificallybinds to osteoclastogenesis-inhibitory factor (OCIF), a process forpreparing the protein, a screening method for a substance which controlsexpression of this protein using this protein, a screening method for asubstance which inhibits or modulates the activity of this protein, ascreening method for the receptor which transmits the activity of thisprotein by binding thereto, a pharmaceutical composition which containsthe substance obtained by these screening methods, an antibody for thesaid protein, and an agent for treating bone metabolism abnormalityusing the antibody.

[0362] Moreover, the present invention provides a DNA encoding a novelprotein (OCIF-binding molecule) which binds toosteoclastogenesis-inhibitory factor (OCIF), a protein which possessesan amino acid sequence encoded by the DNA, a method for preparing theprotein specifically binding to OCIF using said DNA by a geneticengineering technique, and an agent comprising said protein for treatingbone metabolism acatastasia. Furthermore, the present invention providesa screening method for a substance which controls expression of theOCIF-binding molecule, a screening method for a substance which inhibitsor modulates the activity of the OCIF-binding molecule by bindingthereto, a screening method for the receptor which transmits theactivity of the OCIF-binding molecule by binding thereto, and apharmaceutical composition which contains the substance obtained bythese screening methods.

[0363] Still further, the present invention provides a DNA encoding anovel human protein capable of binding to osteoclastogenesis-inhibitoryfactor, OCIF (human OCIF-binding molecule, human OBM), a proteincontaining an amino acid sequence encoded by the DNA, a process forpreparing a protein having characteristics of specifically binding toOCIF and exhibiting a biological activity to support and promote theosteoclast differentiation and maturation by means of geneticengineering technique, and an agent for treating bone metabolismabnormality using the protein. Furthermore, the present inventionprovides a screening method for a substance which controls expression ofthe OCIF-binding molecule, a screening method for a substance whichinhibits or modulates the activity of the OCIF- binding molecule bybinding thereto, a screening method for the receptor which transmits thebiological activity of the OCIF- binding molecule by binding thereto, apharmaceutical composition which contains the substance obtained bythese screening methods, an antibody to human OCIF- binding protein, andan agent for preventing and/or treating bone metabolism abnormalitysymptoms using the antibody.

[0364] In addition, the present invention provides antibodies whichrecognize both antigens (anti-OBM/sOBM antibodies), one is amembrane-bound protein which specifically binds to OCIF (OCIF bindingmolecule; OBM) and the other a soluble OBM (sOBM) which does not have amembrane binding region, a process for preparing the antigen, a methodfor measuring the OBM and sOBM using these antibodies, and an agent forpreventing and/or treating bone metabolism abnormality symptoms usingthe antibody as an effective component.

[0365] The protein and antibody prepared by the process of the presentinvention are useful as medicines and/or reagents for research and testpurposes.

[0366] Description of Deposited Microorganisms

[0367] (1) Name and address of the depository organization to whichmicroorganism was deposited

[0368] Agency of Industrial Science and Technology

[0369] 1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan (postalcode 305)

[0370] Date of deposition to the depository organization

[0371] May 23, 1997

[0372] The deposition number

[0373] FERM BP-5953

[0374] (2) Name and address of the depository organization to whichmicroorganism was deposited

[0375] Agency of Industrial Science and Technology

[0376] 1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan (postalcode 305)

[0377] Date of deposition to the depository organization

[0378] Aug. 13, 1997

[0379] The deposition number

[0380] FERM BP-6058

[0381] (3) Name and address of the depository organization to whichmicroorganism was deposited

[0382] Agency of Industrial Science and Technology

[0383] 1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan (postalcode 305)

[0384] Date of deposition to the depository organization

[0385] Nov. 5, 1997 (Original deposition date)

[0386] The deposition number

[0387] FERM BP-6264

[0388] (4) Name and address of the depository organization to whichmicroorganism was deposited

[0389] Agency of Industrial Science and Technology

[0390] 1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan (postalcode 305)

[0391] Date of deposition to the depository organization

[0392] Nov. 5, 1997 (Original deposition date)

[0393] The deposition number

[0394] FEPM BP-6265

[0395] (5) Name and address of the depository organization to whichmicroorganism was deposited

[0396] Agency of Industrial Science and Technology

[0397] 1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan (postalcode 305)

[0398] Date of deposition to the depository organization

[0399] Nov. 5, 1997 (Original deposition date)

[0400] The deposition number

[0401] FERM BP-6266

1 19 1 316 PRT Mus musculus 1 Met Arg Arg Ala Ser Arg Asp Tyr Gly LysTyr Leu Arg Ser Ser Glu 1 5 10 15 Glu Met Gly Ser Gly Pro Gly Val ProHis Glu Gly Pro Leu His Pro 20 25 30 Ala Pro Ser Ala Pro Ala Pro Ala ProPro Pro Ala Ala Ser Arg Ser 35 40 45 Met Phe Leu Ala Leu Leu Gly Leu GlyLeu Gly Gln Val Val Cys Ser 50 55 60 Ile Ala Leu Phe Leu Tyr Phe Arg AlaGln Met Asp Pro Asn Arg Ile 65 70 75 80 Ser Glu Asp Ser Thr His Cys PheTyr Arg Ile Leu Arg Leu His Glu 85 90 95 Asn Ala Gly Leu Gln Asp Ser ThrLeu Glu Ser Glu Asp Thr Leu Pro 100 105 110 Asp Ser Cys Arg Arg Met LysGln Ala Phe Gln Gly Ala Val Gln Lys 115 120 125 Glu Leu Gln His Ile ValGly Pro Gln Arg Phe Ser Gly Ala Pro Ala 130 135 140 Met Met Glu Gly SerTrp Leu Asp Val Ala Gln Arg Gly Lys Pro Glu 145 150 155 160 Ala Gln ProPhe Ala His Leu Thr Ile Asn Ala Ala Ser Ile Pro Ser 165 170 175 Gly SerHis Lys Val Thr Leu Ser Ser Trp Tyr His Asp Arg Gly Trp 180 185 190 AlaLys Ile Ser Asn Met Thr Leu Ser Asn Gly Lys Leu Arg Val Asn 195 200 205Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His His 210 215220 Glu Thr Ser Gly Ser Val Pro Thr Asp Tyr Leu Gln Leu Met Val Tyr 225230 235 240 Val Val Lys Thr Ser Ile Lys Ile Pro Ser Ser His Asn Leu MetLys 245 250 255 Gly Gly Ser Thr Lys Asn Trp Ser Gly Asn Ser Glu Phe HisPhe Tyr 260 265 270 Ser Ile Asn Val Gly Gly Phe Phe Lys Leu Arg Ala GlyGlu Glu Ile 275 280 285 Ser Ile Gln Val Ser Asn Pro Ser Leu Leu Asp ProAsp Gln Asp Ala 290 295 300 Thr Tyr Phe Gly Ala Phe Lys Val Gln Asp IleAsp 305 310 315 2 1538 DNA Mus musculus 2 gccaggacct ctgtgaaccggtcggggcgg gggccgcctg gccgggagtc tgctcggcgg 60 tgggtggccg aggaagggagagaacgatcg cggagcaggg cgcccgaact ccgggcgccg 120 cgccatgcgc cgggccagccgagactacgg caagtacctg cgcagctcgg aggagatggg 180 cagcggcccc ggcgtcccacacgagggtcc gctgcacccc gcgccttctg caccggctcc 240 ggcgccgcca cccgccgcctcccgctccat gttcctggcc ctcctggggc tgggactggg 300 ccaggtggtc tgcagcatcgctctgttcct gtactttcga gcgcagatgg atcctaacag 360 aatatcagaa gacagcactcactgctttta tagaatcctg agactccatg aaaacgcagg 420 tttgcaggac tcgactctggagagtgaaga cacactacct gactcctgca ggaggatgaa 480 acaagccttt cagggggccgtgcagaagga actgcaacac attgtggggc cacagcgctt 540 ctcaggagct ccagctatgatggaaggctc atggttggat gtggcccagc gaggcaagcc 600 tgaggcccag ccatttgcacacctcaccat caatgctgcc agcatcccat cgggttccca 660 taaagtcact ctgtcctcttggtaccacga tcgaggctgg gccaagatct ctaacatgac 720 gttaagcaac ggaaaactaagggttaacca agatggcttc tattacctgt acgccaacat 780 ttgctttcgg catcatgaaacatcgggaag cgtacctaca gactatcttc agctgatggt 840 gtatgtcgtt aaaaccagcatcaaaatccc aagttctcat aacctgatga aaggagggag 900 cacgaaaaac tggtcgggcaattctgaatt ccacttttat tccataaatg ttgggggatt 960 tttcaagctc cgagctggtgaagaaattag cattcaggtg tccaaccctt ccctgctgga 1020 tccggatcaa gatgcgacgtactttggggc tttcaaagtt caggacatag actgagactc 1080 atttcgtgga acattagcatggatgtccta gatgtttgga aacttcttaa aaaatggatg 1140 atgtctatac atgtgtaagactactaagag acatggccca cggtgtatga aactcacagc 1200 cctctctctt gagcctgtacaggttgtgta tatgtaaagt ccataggtga tgttagattc 1260 atggtgatta cacaacggttttacaatttt gtaatgattt cctagaattg aaccagattg 1320 ggagaggtat tccgatgcttatgaaaaact tacacgtgag ctatggaagg gggtcacagt 1380 ctctgggtct aacccctggacatgtgccac tgagaacctt gaaattaaga ggatgccatg 1440 tcattgcaaa gaaatgatagtgtgaagggt taagttcttt tgaattgtta cattgcgctg 1500 ggacctgcaa ataagttctttttttctaat gaggagag 1538 3 21 DNA Artificial Sequence Description ofArtificial Sequenceprimer 3 aaacgcaaaa aaccagaaag g 21 4 17 DNAArtificial Sequence Description of Artificial Sequenceprimer 4gtaaaacgac ggccagt 17 5 17 DNA Artificial Sequence Description ofArtificial Sequenceprimer 5 caggaaacag ctatgac 17 6 22 DNA ArtificialSequence Description of Artificial Sequenceprimer OBM #8 6 aagccccaaagtacgtcgca tc 22 7 26 DNA Artificial Sequence Description of ArtificialSequenceOBM HF 7 cgaagctttc gagcgcagat ggatcc 26 8 27 DNA ArtificialSequence Description of Artificial SequenceOBM XR 8 cctctagagtctatgtcctg aagtttg 27 9 20 DNA Artificial Sequence Description ofArtificial SequenceOBM3 9 atcagaagac agcactcact 20 10 33 DNA ArtificialSequence Description of Artificial SequenceOBMSalR2 10 ggggtcgacctaggacatcc atgctaatgt tcc 33 11 317 PRT Homo sapiens 11 Met Arg Arg AlaSer Arg Asp Tyr Thr Lys Tyr Leu Arg Gly Ser Glu 1 5 10 15 Glu Met GlyGly Gly Pro Gly Ala Pro His Glu Gly Pro Leu His Ala 20 25 30 Pro Pro ProPro Ala Pro His Gln Pro Pro Ala Ala Ser Arg Ser Met 35 40 45 Phe Val AlaLeu Leu Gly Leu Gly Leu Gly Gln Val Val Cys Ser Val 50 55 60 Ala Leu PhePhe Tyr Phe Arg Ala Gln Met Asp Pro Asn Arg Ile Ser 65 70 75 80 Glu AspGly Thr His Cys Ile Tyr Arg Ile Leu Arg Leu His Glu Asn 85 90 95 Ala AspPhe Gln Asp Thr Thr Leu Glu Ser Gln Asp Thr Lys Leu Ile 100 105 110 ProAsp Ser Cys Arg Arg Ile Lys Gln Ala Phe Gln Gly Ala Val Gln 115 120 125Lys Glu Leu Gln His Ile Val Gly Ser Gln His Ile Arg Ala Glu Lys 130 135140 Ala Met Val Asp Gly Ser Trp Leu Asp Leu Ala Lys Arg Ser Lys Leu 145150 155 160 Glu Ala Gln Pro Phe Ala His Leu Thr Ile Asn Ala Thr Asp IlePro 165 170 175 Ser Gly Ser His Lys Val Ser Leu Ser Ser Trp Tyr His AspArg Gly 180 185 190 Trp Ala Lys Ile Ser Asn Met Thr Phe Ser Asn Gly LysLeu Ile Val 195 200 205 Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn IleCys Phe Arg His 210 215 220 His Glu Thr Ser Gly Asp Leu Ala Thr Glu TyrLeu Gln Leu Met Val 225 230 235 240 Tyr Val Thr Lys Thr Ser Ile Lys IlePro Ser Ser His Thr Leu Met 245 250 255 Lys Gly Gly Ser Thr Lys Tyr TrpSer Gly Asn Ser Glu Phe His Phe 260 265 270 Tyr Ser Ile Asn Val Gly GlyPhe Phe Lys Leu Arg Ser Gly Glu Glu 275 280 285 Ile Ser Ile Glu Val SerAsn Pro Ser Leu Leu Asp Pro Asp Gln Asp 290 295 300 Ala Thr Tyr Phe GlyAla Phe Lys Val Arg Asp Ile Asp 305 310 315 12 954 DNA Homo sapiens 12atgcgccgcg ccagcagaga ctacaccaag tacctgcgtg gctcggagga gatgggcggc 60ggccccggag ccccgcacga gggccccctg cacgccccgc cgccgcctgc gccgcaccag 120ccccctgccg cctcccgctc catgttcgtg gccctcctgg ggctggggct gggccaggtt 180gtctgcagcg tcgccctgtt cttctatttc agagcgcaga tggatcctaa tagaatatca 240gaagatggca ctcactgcat ttatagaatt ttgagactcc atgaaaatgc agattttcaa 300gacacaactc tggagagtca agatacaaaa ttaatacctg attcatgtag gagaattaaa 360caggcctttc aaggagctgt gcaaaaggaa ttacaacata tcgttggatc acagcacatc 420agagcagaga aagcgatggt ggatggctca tggttagatc tggccaagag gagcaagctt 480gaagctcagc cttttgctca tctcactatt aatgccaccg acatcccatc tggttcccat 540aaagtgagtc tgtcctcttg gtaccatgat cggggttggg ccaagatctc caacatgact 600tttagcaatg gaaaactaat agttaatcag gatggctttt attacctgta tgccaacatt 660tgctttcgac atcatgaaac ttcaggagac ctagctacag agtatcttca actaatggtg 720tacgtcacta aaaccagcat caaaatccca agttctcata ccctgatgaa aggaggaagc 780accaagtatt ggtcagggaa ttctgaattc catttttatt ccataaacgt tggtggattt 840tttaagttac ggtctggaga ggaaatcagc atcgaggtct ccaacccctc cttactggat 900ccggatcagg atgcaacata ctttggggct tttaaagttc gagatataga ttga 954 13 27DNA Artificial Sequence Description of Artificial Sequencehuman OBM SF13 ggcgtacgca gagcgcagat ggatcct 27 14 34 DNA Artificial SequenceDescription of Artificial Sequencesynthetic DNA 14 ggggtcgacc atccaggaaatatcataaca ctcc 34 15 951 DNA Mus musculus 15 atgcgccggg ccagccgagactacggcaag tacctgcgca gctcggagga gatgggcagc 60 ggccccggcg tcccacacgagggtccgctg caccccgcgc cttctgcacc ggctccggcg 120 ccgccacccg ccgcctcccgctccatgttc ctggccctcc tggggctggg actgggccag 180 gtggtctgca gcatcgctctgttcctgtac tttcgagcgc agatggatcc taacagaata 240 tcagaagaca gcactcactgcttttataga atcctgagac tccatgaaaa cgcaggtttg 300 caggactcga ctctggagagtgaagacaca ctacctgact cctgcaggag gatgaaacaa 360 gcctttcagg gggccgtgcagaaggaactg caacacattg tggggccaca gcgcttctca 420 ggagctccag ctatgatggaaggctcatgg ttggatgtgg cccagcgagg caagcctgag 480 gcccagccat ttgcacacctcaccatcaat gctgccagca tcccatcggg ttcccataaa 540 gtcactctgt cctcttggtaccacgatcga ggctgggcca agatctctaa catgacgtta 600 agcaacggaa aactaagggttaaccaagat ggcttctatt acctgtacgc caacatttgc 660 tttcggcatc atgaaacatcgggaagcgta cctacagact atcttcagct gatggtgtat 720 gtcgttaaaa ccagcatcaaaatcccaagt tctcataacc tgatgaaagg agggagcacg 780 aaaaactggt cgggcaattctgaattccac ttttattcca taaatgttgg gggatttttc 840 aagctccgag ctggtgaagaaattagcatt caggtgtcca acccttccct gctggatccg 900 gatcaagatg cgacgtactttggggctttc aaagttcagg acatagactg a 951 16 244 PRT Mus musculus 16 AlaGln Met Asp Pro Asn Arg Ile Ser Glu Asp Ser Thr His Cys Phe 1 5 10 15Tyr Arg Ile Leu Arg Leu His Glu Asn Ala Gly Leu Gln Asp Ser Thr 20 25 30Leu Glu Ser Glu Asp Thr Leu Pro Asp Ser Cys Arg Arg Met Lys Gln 35 40 45Ala Phe Gln Gly Ala Val Gln Lys Glu Leu Gln His Ile Val Gly Pro 50 55 60Gln Arg Phe Ser Gly Ala Pro Ala Met Met Glu Gly Ser Trp Leu Asp 65 70 7580 Val Ala Gln Arg Gly Lys Pro Glu Ala Gln Pro Phe Ala His Leu Thr 85 9095 Ile Asn Ala Ala Ser Ile Pro Ser Gly Ser His Lys Val Thr Leu Ser 100105 110 Ser Trp Tyr His Asp Arg Gly Trp Ala Lys Ile Ser Asn Met Thr Leu115 120 125 Ser Asn Gly Lys Leu Arg Val Asn Gln Asp Gly Phe Tyr Tyr LeuTyr 130 135 140 Ala Asn Ile Cys Phe Arg His His Glu Thr Ser Gly Ser ValPro Thr 145 150 155 160 Asp Tyr Leu Gln Leu Met Val Tyr Val Val Lys ThrSer Ile Lys Ile 165 170 175 Pro Ser Ser His Asn Leu Met Lys Gly Gly SerThr Lys Asn Trp Ser 180 185 190 Gly Asn Ser Glu Phe His Phe Tyr Ser IleAsn Val Gly Gly Phe Phe 195 200 205 Lys Leu Arg Ala Gly Glu Glu Ile SerIle Gln Val Ser Asn Pro Ser 210 215 220 Leu Leu Asp Pro Asp Gln Asp AlaThr Tyr Phe Gly Ala Phe Lys Val 225 230 235 240 Gln Asp Ile Asp 17 246PRT Homo sapiens 17 Ala Gln Met Asp Pro Asn Arg Ile Ser Glu Asp Gly ThrHis Cys Ile 1 5 10 15 Tyr Arg Ile Leu Arg Leu His Glu Asn Ala Asp PheGln Asp Thr Thr 20 25 30 Leu Glu Ser Gln Asp Thr Lys Leu Ile Pro Asp SerCys Arg Arg Ile 35 40 45 Lys Gln Ala Phe Gln Gly Ala Val Gln Lys Glu LeuGln His Ile Val 50 55 60 Gly Ser Gln His Ile Arg Ala Glu Lys Ala Met ValAsp Gly Ser Trp 65 70 75 80 Leu Asp Leu Ala Lys Arg Ser Lys Leu Glu AlaGln Pro Phe Ala His 85 90 95 Leu Thr Ile Asn Ala Thr Asp Ile Pro Ser GlySer His Lys Val Ser 100 105 110 Leu Ser Ser Trp Tyr His Asp Arg Gly TrpAla Lys Ile Ser Asn Met 115 120 125 Thr Phe Ser Asn Gly Lys Leu Ile ValAsn Gln Asp Gly Phe Tyr Tyr 130 135 140 Leu Tyr Ala Asn Ile Cys Phe ArgHis His Glu Thr Ser Gly Asp Leu 145 150 155 160 Ala Thr Glu Tyr Leu GlnLeu Met Val Tyr Val Thr Lys Thr Ser Ile 165 170 175 Lys Ile Pro Ser SerHis Thr Leu Met Lys Gly Gly Ser Thr Lys Tyr 180 185 190 Trp Ser Gly AsnSer Glu Phe His Phe Tyr Ser Ile Asn Val Gly Gly 195 200 205 Phe Phe LysLeu Arg Ser Gly Glu Glu Ile Ser Ile Glu Val Ser Asn 210 215 220 Pro SerLeu Leu Asp Pro Asp Gln Asp Ala Thr Tyr Phe Gly Ala Phe 225 230 235 240Lys Val Arg Asp Ile Asp 245 18 735 DNA Mus musculus 18 gcgcagatggatcctaacag aatatcagaa gacagcactc actgctttta tagaatcctg 60 agactccatgaaaacgcagg tttgcaggac tcgactctgg agagtgaaga cacactacct 120 gactcctgcaggaggatgaa acaagccttt cagggggccg tgcagaagga actgcaacac 180 attgtggggccacagcgctt ctcaggagct ccagctatga tggaaggctc atggttggat 240 gtggcccagcgaggcaagcc tgaggcccag ccatttgcac acctcaccat caatgctgcc 300 agcatcccatcgggttccca taaagtcact ctgtcctctt ggtaccacga tcgaggctgg 360 gccaagatctctaacatgac gttaagcaac ggaaaactaa gggttaacca agatggcttc 420 tattacctgtacgccaacat ttgctttcgg catcatgaaa catcgggaag cgtacctaca 480 gactatcttcagctgatggt gtatgtcgtt aaaaccagca tcaaaatccc aagttctcat 540 aacctgatgaaaggagggag cacgaaaaac tggtcgggca attctgaatt ccacttttat 600 tccataaatgttgggggatt tttcaagctc cgagctggtg aagaaattag cattcaggtg 660 tccaacccttccctgctgga tccggatcaa gatgcgacgt actttggggc tttcaaagtt 720 caggacatagactga 735 19 741 DNA Homo sapiens 19 gcgcagatgg atcctaatag aatatcagaagatggcactc actgcattta tagaattttg 60 agactccatg aaaatgcaga ttttcaagacacaactctgg agagtcaaga tacaaaatta 120 atacctgatt catgtaggag aattaaacaggcctttcaag gagctgtgca aaaggaatta 180 caacatatcg ttggatcaca gcacatcagagcagagaaag cgatggtgga tggctcatgg 240 ttagatctgg ccaagaggag caagcttgaagctcagcctt ttgctcatct cactattaat 300 gccaccgaca tcccatctgg ttcccataaagtgagtctgt cctcttggta ccatgatcgg 360 ggttgggcca agatctccaa catgacttttagcaatggaa aactaatagt taatcaggat 420 ggcttttatt acctgtatgc caacatttgctttcgacatc atgaaacttc aggagaccta 480 gctacagagt atcttcaact aatggtgtacgtcactaaaa ccagcatcaa aatcccaagt 540 tctcataccc tgatgaaagg aggaagcaccaagtattggt cagggaattc tgaattccat 600 ttttattcca taaacgttgg tggattttttaagttacggt ctggagagga aatcagcatc 660 gaggtctcca acccctcctt actggatccggatcaggatg caacatactt tggggctttt 720 aaagttcgag atatagattg a 741

What is claimed is:
 1. A novel protein having the followingphysicochemical characteristics and biological activity: (a) affinity:specifically binds to osteoclastogenesis inhibitory factor (OCIF) andexhibits high affinity to OCIF (dissociation constant on cell membrane:Kd=10⁻⁹ M or less); (b) molecular weight: has a molecular weight ofabout 30,000-40,000 or about 40,000±4,000 when measured usingSDS-polyacrylamide electrophoresis under non-reducing conditions and anapparent molecular weight of about 90,000-110,000 when cross-linked to amonomer-type OCIF; and (c) biological activity: exhibits activitysupporting or promoting osteoclasts differentiation and maturation in aco-culture system of the osteoblastic stromal cells and spleen cells inthe presence of such stimulating factors of bone resorption asactive-form vitamin D₃ and parathyroid hormone (PTH).
 2. A process forthe preparation of the protein of claim 1, comprising culturing cells ofan osteoblast-like cell line or stromal cells originating from bonemarrow, in the presence of stimulating factors of bone resorption,preparing a membrane fraction of the cells, solubilizing the membraneprotein using a detergent, and purifying the protein using anOCIF-immobilized affinity column.
 3. A screening method using theprotein of claim 1 for a substance which specifically binds to theprotein of claim 1 and inhibits or modulates the biological activity ofthis protein.
 4. A screening method using the protein of claim 1 for asubstance which specifically binds to the protein of claim 1 andtransmits the biological activity of this protein.
 5. A pharmaceuticalcomposition containing the substance obtained by the screening methoddescribed in claim 3 or
 4. 6. An antibody to the protein of claim
 1. 7.An agent for treating bone metabolism abnormality comprising theantibody of claim 6 as an active component.
 8. A DNA encoding the aminoacid sequence of Sequence ID No. 1 in the Sequence Table.
 9. A DNA whichconsists of the nucleotide sequence of Sequence ID No. 2 in the SequenceTable.
 10. A DNA with one or more nucleotides deleted, substituted,added, or inserted in the nucleotide sequence of Sequence ID No. 2 inthe Sequence Table, and encoding a protein capable of specificallybinding to Osteoclastogenesis Inhibitory Factor and suppressing itsactivity.
 11. A DNA which hybridizes either the DNA of claim 8 or theDNA of claim 10 under relatively mild conditions.
 12. A DNA having 70%or more homology to the DNA of any one of the claims 8-11.
 13. A proteinobtained by the expression of the DNA of any one of the claims 8-12 bygenetic engineering technique.
 14. A protein of claim 13 which exhibitsthe activity to support and promote osteoclast differentiation ormaturation.
 15. The protein having the amino acid sequence of SequenceID No. 1 in the Sequence Table.
 16. A process for producing a protein bygenetic engineering technique using the DNA of any one of the claims8-12.
 17. A screening method for a substance which controls expressionof the protein encoded by the DNA of any one of the claims 8-12.
 18. Ascreening method for a substance which specifically binds to the proteinencoded by the DNA of any one of the claims 8-12 and inhibits ormodulates its biological activity.
 19. A screening method for asubstance which specifically binds to the protein encoded by the DNA ofany one of the claims 8-12 and transmits the biological activity of thisprotein through the binding.
 20. A pharmaceutical composition comprisingthe substance obtained by the screening method described in claims17-19.
 21. A DNA encoding the protein including the amino acid sequenceconsisting of the amino acid residues from No. 76 to No. 316 of SequenceID No. 1 in the Sequence Table, or according to a fragment, analogue, orvariant of the protein.
 22. A DNA encoding the protein including theamino acid sequence consisting of the amino acid residues from No. 72 toNo. 316 of Sequence ID No. 1 in the Sequence Table, or encoding afragment, analogue, or variant of the protein.
 23. A DNA encoding theprotein including the amino acid sequence of Sequence ID No. 1 in theSequence Table, or a fragment, analogue, or variant of the protein. 24.The DNA of any one of the claims 21-23, which encodes a proteinspecifically binding to Osteoclastogenesis Inhibitory Factor, OCIF, andexhibiting the activity of suppressing its biological activity.
 25. ADNA of any one of the claims 21-24 which has a nucleotide sequence ofSequence ID No. 2 in the Sequence Table.
 26. A DNA which hybridizes theDNA of any one of claims 21-25 under relatively mild conditions.
 27. ADNA having 70% or more homology to the DNA of any one of claims 21-26.28. A protein including a polypeptide which is obtained by expression ofthe DNA of any one of the claims 21-27 by genetic engineering technique.29. The protein of claim 28 exhibiting a biological activity to supportand promote the osteoclast differentiation and maturation.
 30. A proteinof claim 28 or 29 including the amino acid sequence of Sequence ID No. 1in the Sequence Table, or a fragment, analogue, or variant of theprotein.
 31. A process for producing a protein by genetic engineeringtechnique using the DNA of any one of the claims 21-27.
 32. A screeningmethod for a substance which controls expression of the protein encodedby the DNA of any one of the claims 21-27.
 33. A screening method for asubstance which specifically binds to the protein encoded by DNA of anyone of the claims 21-27 and inhibits or modulates its biologicalactivity.
 34. A screening method for a substance which specificallybinds to a protein encoded by the DNA of any one of claims 21-27 andtransmits the biological activity of this protein through the binding.35. A pharmaceutical composition comprising the substance obtained bythe screening method of any one of claims 32-34.
 36. A DNA encoding theprotein including the amino acid sequence of Sequence ID No. 11 in theSequence Table, or a fragment, analogue, or variant of the protein. 37.The DNA of claim 36, which encodes a protein specifically binding toOsteoclastogenesis Inhibitory Factor, OCIF, and exhibiting the activityof suppressing its biological activity.
 38. A DNA of any one of claims36-37 which has a nucleotide sequence of Sequence ID No. 12 in theSequence Table.
 39. A DNA which hybridizes to the DNA of any one ofclaims 36-38 under relatively mild conditions.
 40. A DNA having 70% ormore homology to the DNA of any one of claims 36-39.
 41. A proteinincluding a polypeptide which is obtained by expression of the DNA ofany one of the claims 36-40 by genetic engineering technique.
 42. Aprotein of claim 41 which exhibits the activity to support and promotethe differentiation into and the maturation of osteoclasts.
 43. Aprotein of claim 41 or 42 including the amino acid sequence of SequenceID No. 11 in the Sequence Table, or a fragment, analogue, or variant ofthe protein.
 44. A process for producing a protein by a geneticengineering technique using the DNA of any one of the claims 36-40. 45.A screening method for a substance which controls expression of theprotein encoded by the DNA of any one of the claims 36-40.
 46. Ascreening method for a substance which specifically binds to the proteinencoded by the DNA of any one of claims 36-40 and inhibits or modulatesits biological activity.
 47. A screening method for a substance whichspecifically binds to the protein encoded by the DNA of any one ofclaims 36-40 and transmits the biological activity of this proteinthrough the binding.
 48. A pharmaceutical composition comprising thesubstance obtained by the screening method any one of claims 45-47. 49.An antibody which recognizes a membrane-bound protein molecule (OBM)which binds specifically to Osteoclastogenesis Inhibitory Factor (OCIF)and/or a soluble OBM (sOBM), which is deficient in the transmembraneregion.
 50. The antibody of claim 49 which recognizes mouse OBM and/ormouse sOBM.
 51. The antibody of claim 49 which recognizes human OBMand/or human sOBM.
 52. The antibody of claim 49 which is a polyclonalantibody.
 53. The antibody of claim 49 which is a monoclonal antibody.54. A monoclonal antibody of claim 53 which has cross-reactivity tohuman OBM, human sOBM, mouse OBM, and mouse sOBM.
 55. The monoclonalantibody of claim 53 which possesses following characteristics: a)molecular weight: about 150,000, b) subclass: IgG₁ or IgG₂, and c) lightchain: κ chain
 56. A process for preparing a polyclonal antibodycomprising immunizing an animal with mouse or human OBM or sOBM andpurifying from blood of the animal.
 57. A hybridoma which produces themonoclonal antibody of any one of claims 53-55.
 58. The hybridomas withdeposition numbers FERM BP-6264, FERM BP-6265, or FERM BP-6266.
 59. Aprocess for preparing a monoclonal antibody comprising cultivating thehybridoma of claim 57 or 58 to produce a monoclonal antibody in theculture broth and purifying and recovering the monoclonal antibody fromthe culture broth.
 60. A method for measuring OBM, comprising using theantibody of any one of the claims 49-55.
 61. A method for measuringsOBM, comprising using the antibody of any one of claims 49-55.
 62. Amethod for measuring OBM or sOBM, comprising using the antibody of anyone of claims 49-55 as an solid phase antibody or an enzyme-labeledantibody.
 63. A pharmaceutical composition containing the antibody ofany one of claims 49-55 as an active component.
 64. An agent forpreventing or treating bone metabolism diseases comprising the antibodyof any one of claims 49-57 as an active component.